Effector Granules in Human T Lymphocytes - American Chemical

ARTICLE pubs.acs.org/jpr

Effector Granules in Human T Lymphocytes: Proteomic Evidence for Two Distinct Species of Cytotoxic Effector Vesicles Hendrik Schmidt,† Christoph Gelhaus,‡ Melanie Nebendahl,† Marcus Lettau,† Ralph Lucius,§ Matthias Leippe,‡ Dietrich Kabelitz,† and Ottmar Janssen*,† †

Institute of Immunology, ‡Zoological Institute, and §Institute of Anatomy, Christian-Albrechts-University, Kiel, Germany

bS Supporting Information ABSTRACT: Cytotoxic T cells mobilize effector proteins from prestored lysosomal compartments. Since different activation signals result in alternative routes of target cell killing, utilizing either FasL or the granzyme B/perforin pathway, the existence of distinct forms of effector granules was recently suggested. Applying a protocol for the separation of intact organelles from activated T lymphoblasts, we noticed that FasL-associated secretory lysosomes (SL) segregate from vesicles containing larger amounts of granzymes and granulysin. We previously analyzed the proteome of secretory lysosomes from NK and T cells and now describe the proteome of granzyme-containing vesicles. Moreover, intact FasL-associated SL and granzymecontaining vesicles were compared by electron microscopy and respective extracts were characterized by Western blotting. With the present report, we provide a comprehensive proteome map of granzyme-containing granules and unequivocally demonstrate that T lymphoblasts contain at least two distinct types of effector vesicles. Moreover, the overall protein content of the two vesicle populations was compared by 2D difference gel electrophoresis. Interestingly, the observed differences in protein distribution were not restricted to effector proteins but also applied to cytoskeleton-associated elements that could argue for a differential transport or initiation of degranulation. To our knowledge, this is the first comprehensive description of distinct effector granules in T cells. KEYWORDS: 2D-electrophoresis, 2D-DIGE, proteome analysis, mass spectrometry, organelles, secretory lysosomes, T lymphocytes, FasL, granzymes

’ INTRODUCTION Cytotoxic T cells (CTL) lyse their target cells through the surface expression of Fas ligand (FasL) and/or the release of soluble mediators such as perforin, granzymes and granulysin. According to a widely accepted model, lytic effector proteins are stored in so-called secretory lysosomes (SL), which are bifunctional organelles that combine degradative and secretory properties.1,2 This model was based on the observation that intracellular FasL, which is regarded as the marker protein for SL, colocalizes with other lysosomal markers such as LAMP-1 (CD107a), LAMP-3 (CD63), and also with effector proteins including perforin and granzymes.3,4 Upon target cell contact, degranulation of a single type of organelles would ensure a coordinated surface expression of FasL and the simultaneous delivery of effector molecules into the immunological synapse. In recent years, however, a more complex regulation of FasL expression has been shown for human and murine T cells.5,6 Upon activation, we observed a biphasic FasL surface appearance on human T and NK cells and showed that phase one (minutes after stimulation) relies on an actin-dependent mobilization of preformed FasL whereas phase two (hours after stimulation) requires de novo protein synthesis.5 These two waves of FasL r 2011 American Chemical Society

appearance were also observed in murine CTLs by He and Ostergaard.6 They suggested that the FasL phase one release results in a rapid target cell elimination in response to low concentrations of antigen. In contrast, the second wave depends on newly synthesized FasL, requires higher antigen concentrations and mediates extensive Fas-dependent bystander killing in the absence of detectable degranulation. Based on the observation that FasL was found in vesicles distinct from cytolytic granules, it was concluded that the lytic mechanisms are fully independent with respect to localization and regulation of the individual cytotoxic components.6,7 A differential regulation of FasL and cytotoxic granules was then supported by Kassahn and colleagues, who demonstrated that the activation-induced exocytosis of FasL had other requirements than the surface appearance of the granule (lysosome) markers CD63 (LAMP-3) and CD107a (LAMP-1).8 In fact, the presence of two differentially regulated CTL effector pathways had already been suggested in 1998, when Kessler and colleagues reported that a weak TCR signal would still lead to Fas-dependent cytotoxicity whereas perforin-dependent killing required a stronger TCR signal.9 Received: September 21, 2010 Published: January 19, 2011 1603

dx.doi.org/10.1021/pr100967v | J. Proteome Res. 2011, 10, 1603–1620

Journal of Proteome Research We have recently provided a protocol to enrich FasL-associated secretory lysosomes from lymphocytes.10 When we characterized individual fractions of a given organelle preparation by Western blotting, we noticed that not all cytotoxic effector molecules display a comparable distribution pattern. As described, FasL-associated secretory lysosomes are reproducibly enriched in gradient fraction 2 of T and NK cells.10-12 In contrast, intact vesicles containing high amounts of granzymes A and B and granulysin separated from SL and were found in fraction 6 of the applied gradient.10 Having analyzed the luminal proteome of FasL-containing vesicles in NK cells11 and T cell blasts,12 we now determined the protein content of fraction 6 vesicles. Furthermore, we directly compared the proteome of the FasL-associated SL compartment and the granzyme B-rich compartment using 2D difference gel electrophoresis. Based on these 2D-DIGE results, we verified several of the observed differences by Western blotting. In conclusion, we provide the first biochemical evidence for the coexistence of two types of cytotoxic effector granules in activated lymphocytes.

’ MATERIAL AND METHODS Cells

Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations by Ficoll density gradient centrifugation. T cells were further purified by MACS Cell Isolation Kits (Miltenyi, Bergisch Gladbach, Germany). To obtain activated T lymphoblasts, the cells were stimulated with phytohemagglutinin A (PHA, 0.5 μg/mL; Remel, Lenexa, KS) and further expanded in the presence of recombinant human interleukin 2 as described.13 Cells were kept in RPMI1640 with HEPES and L-glutamine, 5% fetal calf serum and antibiotics at 37 °C in a humidified atmosphere with 5% CO2. Subcellular Fractionation

Organelle fractions were separated following the recently described procedure.10 In brief, 4 108 cells were carefully disrupted in a dounce glass homogenizer and organelles where enriched by differential centrifugation. The enriched organelles were placed on a nonionic, low osmotic discontinuous density gradient with 27, 22.5, 19, 16, 12, and 8% OptiPrep (Sigma, Deisenhofen, Germany) and centrifuged at 150 000 g for 5 h in a SW60Ti swing-out rotor (Beckman Coulter, Krefeld, Germany). Obtained organelles were washed and lysed for further downstream analyses. Confocal Microscopy

PHA-stimulated lymphocytes were fixed with 3% paraformaldehyde, permeabilized with Triton X-100 and stained with antigranulysin (R&D Systems, Minneapolis, MN) and anti-goat AlexaFluor555 (Invitrogen) and, after extensive washes, with AlexaFluor647-conjugated anti-granzyme B (clone GB11, BD Biosciences, Franklin Lakes, NJ) and FITC-labeled antigranzyme A (clone GrA-11, Immunotools, Friesoythe, Germany). Alternatively, samples were stained with anti-FasL mAb NOK-1 (BD Biosciences) and AlexaFluor488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany), and subsequently with AlexaFluor647-labeled antigranzyme B mAb. Stained samples were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed on a LSM 510 Meta laser scanning microscope (Carl Zeiss, Jena, Germany). Images were acquired via scanning through the x-y-plane with 63 objective lenses. Laser intensity and detectors were adjusted to a uniformly

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negative signal of the control samples stained with control IgG and second step antibodies. Western Blot Analysis

For Western blotting, the protein content of individual samples was determined using the Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Five μg of protein per fraction were separated on Bis-Tris gels (4-12%, Invitrogen). After transfer to nitrocellulose membranes and blocking with 5% bovine serum albumin or fat-free dry milk in TBST following the individual manufacturer’s recommendations, the fractions were analyzed using anti-FasL mAb (clone G-247.4, BD Biosciences), antiCD63 clone MEM-259 (Acris Antibodies, Herford, Germany), anti-LAMP-1 mAb clone 25 (BD Biosciences), anti-cathepsin D clone CTD-19 (Sigma), anti-Vti-1b clone 7 (BD Biosciences), anti-pan-cadherin clone ab22744 (Abcam, Cambridge, UK), anticytochrome oxidase IV (CoxIV) clone 10G8D12C12 (MitoScience, Eugene, OR), anti-Bip/Grp78 clone 40 (BD Biosciences), antigranzyme B clone 2C5/F5 (BD Biosciences) and anti-granulysin polyclonal antibodies (R&D Systems, Minneapolis, MN, USA). The following antibodies were used for verification at later stages: anti-β-actin clone AC-15 (Sigma), anti-DPPIV clone 222113 (R&D Systems), anti-Ezrin/Radixin/Moesin (ERM) polyclonal rabbit sera (Cell Signaling, Danvers, MA), anti-granzyme A clone YMH01 (R&D Systems), antimyosin IIA polyclonal sera (Abcam), anti-dynamin I/II polyclonal rabbit sera (Cell Signaling) and anti-flotilin-1 clone 18 (BD Biosciences) with respective horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Munich, Germany). For reprobing, membranes were incubated in stripping solution for 25 min at 56 °C. ECL detection reagents were used for chemiluminescence detection using Hyper Film (GE Healthcare). Transmission Electron Microscopy

Enriched organelles of fractions 2 and 6 were fixed with 3% paraformaldehyde in PBS at 4 °C overnight, washed in PBS, postfixed in 2% OsO4, dehydrated in ethanol, and embedded in Araldite (Sigma). Ultrathin sections were mounted on Formvarcoated grids and double-stained with a saturated solution of uranyl acetate in 70% methanol and lead citrate. The grids were examined with a Zeiss EM 900 transmission electron microscope equipped with a digital camera system. 2D-Electrophoresis, Image Analysis and Spot Picking

For 2D electrophoresis, pellets of fraction 6 isolates were lyzed on ice for 30 min as described before.11 A total amount of 250 μg of protein was mixed with rehydration buffer and applied by cuploading onto 24 cm IPG strips (nonlinear, pH 3-11). Isoelectric focusing (IEF) was carried out following the described standardized scheme.10 The second dimension was performed on large format (26 20 cm) 12.5% polyacrylamide gels. Gels were stained with Flamingo Pink (Bio Rad, Munich, Germany), mounted on a nonbacked gel frame, scanned on a Typhoon Trio (GE Healthcare) and analyzed using Image Master 6.0 (GE Healthcare). Selected spots were picked with a 2 mm picking head. The picked gels were again scanned to verify the correct location of the punched spots. For 2D difference gel electrophoresis (2D-DIGE), 50 μg of the fraction 2 and 6 extracts were labeled with Cy3 or Cy5, respectively (CyDyes, GE Healthcare). A pool of all samples was labeled with Cy2 and served as an internal standard for biological variance analysis (BVA). After quenching with lysine, the differentially labeled samples were pooled and applied to 2D-electrophoresis 1604

dx.doi.org/10.1021/pr100967v |J. Proteome Res. 2011, 10, 1603–1620

Journal of Proteome Research

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Figure 1. Colocalization of cytotoxic proteins in T cell blasts. PHA stimulated lymphocytes were fixed and stained as described in Materials and Methods for (A) granzyme B and FasL or for (B) granzyme B, granzyme A and granulysin.

Figure 3. Organelle characteristics analyzed by transmission electron microscopical inspection. Comparison of organelles obtained in fractions 2 (A,B) and 6 (C,D). Enriched organelles from lymphoblasts were fixed and embedded in Araldite. Overview pictures are given in (A) and (C), magnified areas are shown in (B) and (D).

Figure 2. Cytotoxic proteins segregate in different fractions. Equal amounts of whole cell lysate (WL), enriched organelles (EO) and six fractions collected after density gradient centrifugation of PHA blasts were separated and transferred to NC. Blots were probed with antibodies against the indicated proteins. Note that the two distinct granulysin bands represent the unprocessed proform and the processed active form of 15 and 9 kDa, respectively.

as described above. Image analysis of the scanned fluorescent gels using differential in-gel analysis (DIA) included in the DeCyder 6.5 software (GE Healthcare). For BVA, Cy2 gel images were merged; spot boundaries were detected followed by a normalization of the spot volumes revealing differential spots with a t test filter set to 0.05.

entries) updated frequently with MASCOT using the following parameters: the modification on cysteine residues by carbamidomethylation was set as obligate, methionine oxidation was considered as a potential modification; the maximum number of missed tryptic cleavages was one; the monoisotopic masses were considered and the mass tolerance was set to (50 ppm, and the fragment-ion mass tolerance was set to 0.2 Da (MS/MS). A protein was accepted to be identified when the total protein score reached or exceeded the MASCOT score threshold (g66 with a probability of identification greater 95%). Repetitive searches against a randomized decoy database (http://www.matrixscience. com/help/decoy_help.html) using the decoy.pl script and identical search parameters let to a false-positive rate of 2.8%. Classification of Proteins

The classification of individual proteins according to their localization was based on the Uni-Prot knowledge base, the ihop database14 and the iProXpress database15 from the “Protein Information Resource” (GUMC, Washington, D.C.).

In-Gel Tryptic Digestion and Mass Spectrometry

Mass spectrometric analyses were performed on a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA) with minor modifications as described before.11 Ten microliters sequencing grade porcine trypsin (Serva, Heidelberg, Germany) at a concentration of 10 ng/μL in 5 mM ammonium bicarbonate were applied per spot for in-gel proteolysis. For the parent runs, mass spectra were internally calibrated with the masses of trypsin’s autolysis products with 25 ppm mass tolerance. Five precursors per spectrum were selected from the most intense peaks and submitted to MSMS analyses. The updated default calibration was used for MSMS measurements. Masses were searched in NCBI nonredundant database (10/10/2008, 217 337

’ RESULTS Localization of Lysosome-Associated Effector Proteins in T Cell Blasts

In order to visualize the presence of secretory lysosomes in human T cell blasts, most laboratories routinely employ confocal microscopy. In general, costaining for FasL and other characteristic lysosomal markers including cathepsin D, LAMP-1 or CD63 reveals a high degree of colocalization.3,4,13,16-18 As depicted in Figure 1A, confocal imaging in fact suggested a FasL colocalization with effector proteins such as granzyme B in larger vesicular structures. Using the same settings for detection, a colocalization of granzyme 1605



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Figure 4. Proteomic map of fraction 6 granules. Lysates of fraction 6 organelles were separated on pH 3-11NL IPG strips in the first dimension and on 12.5% Tris-glycin gels in the second dimension. Flamingo Pink staining was used for protein visualization. Annotated numbers of 379 identified spots refer to Table S1 (Supporting Information). Enlarged quadrants of the gel image are available as Supporting Information (Figure S6A-D).

B with granzyme A and granulysin, two other effector proteins released by degranulation, is shown in Figure 1B. In view of the recent discussion on the existence of distinct lysosomal compartments for degranulation and transport of FasL,6-8 and based on the biochemical evidence that we had obtained earlier, we decided to address this issue in more detail and to perform a thorough biochemical analysis of enriched organelles from T cell blasts. Western Blot Analyses Suggest the Existence of Distinct Effector Granules

FasL serves as an accepted marker protein for secretory lysosomes (SL) from T and NK cells.16 As detected by Western blotting, FasL is reproducibly enriched in fraction 2 vesicles of a standardized Iodixanol gradient.10 This fraction is further characterized by an enrichment of lysosomal membrane proteins (LAMP-1, CD63) and lysosomal hydrolases such as cathepsin D (Figure 2). Assuming that fraction 2 vesicles represent FasLcontaining SL, we recently compiled the luminal proteome of secretory lysosomes from NK cells11 and activated T cells.12 Interestingly, perforin and granzymes A and B were not always detected in the SL fraction at comparable levels. As shown in detail for different NK cell populations, these effector proteins were unevenly distributed in isolates from individual cell populations indicating potential differences in granular distribution and effector function.11 As demonstrated in Figure 2, Western blotting of individual fractions from T cell blasts confirmed that granzyme B and granulysin were significantly enriched in fraction 6 and not in fraction 2 (as initially anticipated). Moreover, granulysin showed a particularly interesting fractionation pattern with the 15 kDa precursor form being enriched in fraction 2 and the processed active 9 kDa form being significantly enriched in fraction 6. To test whether the two fractions were devoid of potential organelle contaminants, we used antibodies against

marker proteins for plasma membrane (cadherin), mitochondria (CoxIV) and endoplasmic reticulum (Bip/Grp78). Here, we only detected some minor contaminations with ER proteins in fraction 2. Morphological Characteristics of Organelles in Different Fractions

For the description of the organelle enrichment protocol, we had already contrasted organelles obtained in fraction 2 to those in fraction 6 of our gradient.10 We also compared intact fraction 2 vesicles and organelles found in fraction 5 (mitochondria).12 To verify these results and to highlight the differences between the two types of vesicles containing cytotoxic effector proteins, we analyzed additional samples by electron microscopy employing a slightly modified fixation protocol. As shown in Figure 3, fraction 2 vesicles show a maximum diameter of 700 nm. Structurally, these vesicles display a homogeneous appearance with an dense lumen surrounded by a double membrane. Of note, only in fraction 2 preparations, we also infrequently observed organelles displaying a multivesicular structure (Figure S1A, Supporting Information). In contrast, fraction 6 contained a homogeneous organelle population with the characteristic morphology that we described before.10 With a diameter of up to 300 nm, these membrane-surrounded granules were mostly smaller than fraction 2 vesicles. They displayed electron-dense cores and in most cases a characteristic inner circular, membrane-surrounded structure that suggested a polar content distribution (Figure 3D, Figure S1B, Supporting Information). Proteome of Fraction 6 Vesicles

To further characterize fraction 6 vesicles, we deciphered the proteome of respective isolates from human T cell blasts. From two preparative gels, we identified 379 spots representing 240 individual proteins. According to the subsequent database analyses, 67% of the identified proteins had been associated 1606



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Table 1. Proteome of Iodixanol Gradient Fraction 6 Vesicles from T Lymphoblastsa spot no. replicates

Uni-Prot no.

MW, Da

pI

% seq. MASCOT coverage

function

subcellular localization

protein name

acc. no. gi|77404397 gi|157502193

Q7KZF4 102618 Q9UNM6 43203

6.7 5.5

400 265

36 19

transcription protein binding

ER,ME,PL ME

gi|4504505 gi|12654583 gi|49168620 gi|5031599 gi|119618319 gi|5031593 gi|15277503 gi|5031571 gi|5031573 gi|29029550 gi|178413 gi|1419374 gi|18645167 gi|4757810 gi|4885079 gi|55959356

P51659 80092 P05388 34424 Q99798 86155 O15144 34426 O15145 19108 O15511 16367 P60709 40536 P61160 45017 P61158 47797 Q9NZK5 59180 P35475 72988 O00754 111834 Q8TBV2 38780 P25705 59828 P36542 32917 P24539 22318

9.0 5.4 7.1 6.8 8.7 5.5 5.6 6.3 5.6 7.8 9.3 6.7 7.6 9.2 9.3 9.3

341 378 447 504 150 102 641 552 536 280 203 206 439 593 326 91

25 41 22 59 30 37 53 43 36 27 15 9 56 50 28 23

multifuntional ribonucleoprotein hydrolase trafficking trafficking trafficking trafficking trafficking trafficking hydrolase hydrolase hydrolase vesicle fusion ion transport ion transport ion transport

ER,ME,EN,PE ER,EN,ME EX,SY,MT,NU EN,ME,PL EN,ME,PL,ER EN,ME ER,ME,EX,PL,SY ER,EN,ME ER,EN,ME secreted LY LY,ME ME ER,LY,NG,SY,MT LY;ME,MT LY,ME,MT

gi|51479152

O75947

15820

6.6

127

33

ion transport

ER,ME,NG,SY,MT

gi|6730518 gi|2554776 gi|30749651 gi|9836652 gi|14602585

P61769 11767 P08236 70870 P07686 58480 Q9HDC9 47887 P11586 102152

5.8 6.3 6.3 5.8 6.8

90 301 303 381 533

29 27 32 39 48

immunity hydrolase hydrolase biosynthesis multifunctional

EN,ME,PL LY,EX LY,ME,NG ME EN,ME,PL,MT

gi|4557014 gi|157833437 gi|4503143

P04040 P07858 P07339

59947 36000 45037

6.9 5.8 6.1

417 274 295

37 19 33

oxidoreductase hydrolase hydrolase

44 315

1 1

96 349 73 404 537 550 282 309 233 170 119 113 363 218 400 492

1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1

501

1

614 91 224 236 53

2 1 1 1 1

166 422 437

1 1 1

100 kDa coactivator 26S proteasome non-ATPase regulatory subunit 13 3-hydroxyacyl-CoA dehydrogenase 60S acidic ribosomal protein P0 aconitate hydratase, mitochondrial actin related protein 2/3 complex subunit 2 actin related protein 2/3 complex subunit 3 actin related protein 2/3 complex subunit 5 actin, beta actin-related protein 2 actin-related protein 3 Adenosine deaminase CECR2 alpha-L-iduronidase alpha-mannosidase annexin A2 ATP synthase subunit alpha, mitochondrial ATP synthase subunit gamma ATP synthase, Hþ transporting, F0 complex, subunit B1 ATP synthase, Hþ transporting, F0 complex, subunit D beta-2-microglobulin beta-glucuronidase beta-hexosaminidase subunit beta BSCv protein C-1-tetrahydrofolate synthase, cytoplasmic catalase cathepsin B cathepsin D preproprotein

481

1

cathepsin S

gi|30749675

P25774

24491

7.6

369

47

hydrolase

ER,LY,EN,ME LY,ME,NG LY,ME,NG,EX, MT LY

532 465

1 1

cathepsin W chromosome 14 open reading frame 166

gi|119594869 gi|119586064

P56202 Q9Y224

30154 26652

8.6 6.7

76 392

36 62

hydrolase protein binding

LG ME,EN,ER,CY,NU

186

1

chromosome 22 open reading frame 28

gi|12652799

Q9Y3I0

55722

6.8

260

36

protein binding

EN,ME

544

1

cofilin-1

gi|5031635

P23528

18719

8.2

93

34

actin binding

ER,ME,EX,MT, NU

175

1

coronin, actin binding protein, 1A

gi|5902134

P31146

51678

6.3

203

26

actin binding

LY,PL

249

2

Cytochrome b-c1 complex subunit 1, mitochondrial

gi|515634

P31930

53270

5.9

693

57

electron transport

ME,PL,MT

466

1

Cytochrome b-c1 complex subunit 11

gi|45768728

P47985

29958

8.6

377

32

electron transport

ER,MT,ME

292

2

Cytochrome b-c1 complex subunit 2, mitochondrial

gi|50592988

P22695

48584

8.7

748

49

electron transport

ER,ME,MT

68 190 206 29 307 383 59 163 252 283 448 243 146 217

1 1 1 1 1 1 2 1 1 2 1 1 1 1

gi|4826686 gi|83753870 gi|15080291 gi|27574040 gi|7706495 gi|27597059 gi|56549121 gi|2529707 gi|39644794 gi|704416 gi|192987144 gi|203282367 gi|7657069 gi|4759034

Q92499 P09622 Q9UHL4 P27487 Q9UBS4 Q8WXX5 P50570 Q9H4M9 P26641 P49411 P30040 P06733 Q96HE7 P62495

83349 50656 54749 85008 40774 30062 98345 60722 50156 49851 27220 47350 55213 49228

6.8 6.5 5.9 5.7 5.8 5.6 7.0 6.5 6.3 7.7 7.1 7.0 5.5 5.5

401 485 185 344 446 477 98 454 302 621 158 592 356 248

37 51 18 15 40 42 16 39 29 53 28 69 32 22

protein binding oxidoreductase hydrolase hydrolase chaperone chaperone motor protein trafficking protein binding biosynthesis protein transport multifunctional oxidoreductase biosynthesis

ME ME,MT ME,LY,NG ER,LY,EN,ME,EX ER,ME,PL ME EN ER,LY,EN,EX,PL ME LY,ME,PL,MT ER,ME,PL ME,EX,SY,MT ME,ER ER,EN

57

1

DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 dihydrolipoyl dehydrogenase, mitochondrial dipeptidyl peptidase 7 dipeptidyl peptidase IV (CD26) DnaJ homologue subfamily B member 11 DnaJ homologue, subfamily C, member 9 dynamin 2 EH-domain containing 1, isoform CRA_a elongation factor 1-gamma elongation factor Tu endoplasmic reticulum protein ERp29 enolase 1 ERO1-like [Homo sapiens] eukaryotic peptide chain release factor subunit 1 eukaryotic translation elongation factor 2

gi|4503483

P13639

96246

6.4

598

34

biosynthesis

ER,EN,ME,EX

1607



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Table 1. Continued spot no. replicates

protein name

acc. no.

Uni-Prot no.

MW, Da

pI


function


88 348 397

1 1 1

ezrin F-actin-capping protein subunit alpha-1 F-actin-capping protein subunit beta

gi|46249758 gi|5453597 gi|119615295

P15311 P52907 P47756

69313 33057 30226

5.9 5.5 5.9

461 470 238

45 47 48

actin binding actin binding actin binding

ME,EX,MT ER,EN,ME ME,EX

521 242 322 270 144 472 443 370 314 49 215

1 1 1 1 1 1 1 1 1 1 1

gi|110591399 gi|5031699 gi|4557305 gi|119590499 gi|119584846 gi|178045 gi|178045 gi|4503987 gi|63252913 gi|119594451 gi|20151189

P02792 O75955 P04075 P07954 P16278 P63261 P63261 Q92820 P40121 Q14697 P00367

19933 47554 39851 46555 40868 26147 26147 36340 38760 104930 56315

5.5 7.1 8.3 6.9 8.8 5.7 5.7 6.7 5.8 5.9 6.7

226 577 331 301 148 389 467 365 237 376 641

32 65 42 33 22 38 51 28 15 31 66

transport protein binding Lyase lyase hydrolase trafficking trafficking hydrolase actin binding hydrolase oxidoreductase

ME,EX LY,ME,EX ME,EX,SY,MT EN,SY,MT LY,ME,EX ME,EX,PL,SY ME,EX,PL,SY LY,ME,NG,PL ME, NU ER,ME,PL ER,ME,PL,MT

350

1

gi|31645

P04406

36202

8.3

397

36

glycolysis

452 430 271

2 1 1

ferritin light chain flotillin 1 fructose-bisphosphate aldolase A fumarate hydratase, isoform CRA_d galactosidase, beta 1 gamma-actin gamma-actin gamma-glutamyl hydrolase precursor gelsolin-like capping protein glucosidase II subunit alpha glutamate dehydrogenase 1, mitochondrial glyceraldehyde-3-phosphate dehydrogenase granzyme A granzyme B growth-inhibiting protein 1

gi|33357774 gi|13096242 gi|34452679

P12544 P10144 O14773

26328 25894 34670

9.1 9.6 5.7

604 119 464

70 18 27

cytolysis cytolysis protease

471 325

2 1

gi|5107637 gi|119585458

P62826 P04899

24519 20295

7.0 5.5

493 305

47 80

transport signal transduction

LY,ME,NG,EX, PL,SY,MT LG LG LY,ME,NG, PL,MT ME,EX EX

366

1

gi|306785

P62879

38061

5.8

382

32

signal transduction

EN,ME,EX,SY,MT

256 90 108

1 1 1

gi|219588 gi|16507237 gi|5729877

P31689 P11021 P11142

45590 72402 71082

6.7 5.1 5.4

512 692 504

44 48 38

chaperone chaperone chaperone

106 40 64 610 316 126 27

1 1 1 1 1 1 1

gi|24234688 gi|15010550 gi|119602173 gi|9256890 gi|460771 gi|24308207 gi|31621305

P38646 73890 P14625 90309 P07900 57868 P69905 30476 Q15365 38015 Q8N1G4 64004 P42704 159060

6.0 4.7 4.9 8.9 6.7 8.6 5.8

466 325 169 157 307 114 118

33 26 14 28 47 18 7

chaperone chaperone chaperone transport DNA/RNA binding protein binding transport

ER,ME ER,ME,EX,PL,MT LY,ME,NG,EX, PL,SY,MT ME,EX,PL,SY,MT ER,ME,PL ME,NG,MT SY,ER,MT ME,NU,CY ME ME,MT

376 280 98 192 39 371 296 313

1 1 1 1 1 1 1 1

gi|62897717 gi|13111975 gi|114159823 gi|4826940 gi|1097308 gi|49168580 gi|20987224 gi|17834080

P00338 P11117 P10253 P42785 Q14764 P40925 P53582 O96008

36951 48685 106142 56263 100135 35993 30919 33904

7.6 6.1 5.6 6.8 5.3 8.9 6.2 6.8

274 106 126 101 365 880 212 229

31 15 9 15 26 69 32 27

metabolism degradation hydrolase degradation protein transport oxidoreductase hydrolase ion transport

ME,EX,SY LY,ME LY,NG,EX LY,ME,NG ME ME,EX,PL,SY,MT EN MT,ME

74 311 605

1 1 1

gi|516764 gi|23879 gi|11496277

Q16891 P28482 Q9UHA4

9830 40794 13671

5.7 6.7 6.7

463 143 68

25 37 30

protein binding signal transduction protein binding

ME,MT ME,PL LY

94 62 601

1 1 1

gi|119625804 gi|12667788 gi|189011548

P26038 P35579 Q13510

66678 227646 45087

5.9 5.5 7.5

677 481 86

44 14 11

structure motor protein hydrolase

EN,ME,EX,PL,MT EN,ME,EX,PL,MT LY

500

1

gi|4758774

O96000

21048

8.7

148

51

electron transport

MT,ME

455

1

gi|4758788

O75489

30337

7.0

623

53

electron transport

ER,MT,ME

95

1

gi|21411235

P28331

80415

5.8

582

45

electron transport

ER,ME,MT

102 554 360 426

1 1 1 1

gi|10257494 gi|66392203 gi|8923427 gi|4506031

P46459 P22392 Q9NX40 P50897

83113 30346 27780 34627

6.4 9.1 7.0 6.1

293 107 249 522

23 35 34 29

protein transport metabolism unknown degradation

EN,ME,SY ER,ME,SY,MT EN LY,ME,NG

GTP-binding nuclear protein Ran guanine nucleotide-binding protein G(i), alpha-2 subunit guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 heat shock 40 kDa protein 4 heat shock 70 kDa protein 5 heat shock 70 kDa protein 8 isoform 1 heat shock 70 kDa protein 9 heat shock protein 90 kDa beta member 1 heat shock protein HSP 90-alpha hemoglobin subunit alpha hnRNP-E1 leucine rich repeat containing 47 leucine-rich PPR motif-containing protein L-lactate dehydrogenase lysosomal acid phosphatase lysosomal alpha-glucosidase lysosomal Pro-X carboxypeptidase Major Vault Protein malate dehydrogenase methionyl aminopeptidase 1 mitochondrial import receptor subunit TOM40 homologue mitochondrial inner membrane protein mitogen-activated protein kinase 1 mitogen-activated protein kinase scaffold protein 1 moesin myosin 9 N-acylsphingosine amidohydrolase 1 isoform a preproprotein NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22 kDa NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30 kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial N-ethylmaleimide-sensitive factor NME1-NME2 protein OCIA domain-containing protein 1 palmitoyl-protein thioesterase 1

1608



ARTICLE

Table 1. Continued spot no. replicates

protein name

acc. no.

Uni-Prot no.

MW, Da

pI



function

566 541 136 504

1 1 1 1

peptidyl-prolyl cis-trans isomerase A peptidyl-prolyl cis-trans isomerase B perforin-1 peroxiredoxin 1

gi|1633054 gi|1310882 gi|40254808 gi|55959887

P62937 P23284 P14222 Q06830

18098 19705 62479 19135

7.8 9.2 8.1 6.4

287 104 67 336

68 36 7 49

isomerase isomerase cytolysis oxidoreductase

496

1

peroxiredoxin 2

gi|1617118

P32119

18486

5.2

69

35

oxidoreductase

489 117 453 590 135 251 168

1 1 1 2 1 1 1

peroxiredoxin 3 phenylalanyl-tRNA synthetase beta chain phosphoglycerate mutase 1 profilin-1 programmed cell death 8 isoform 2 proliferation-associated protein 2G4 prolyl 4-hydroxylase, beta subunit

gi|119569783 gi|7768938 gi|38566176 gi|157838211 gi|22202629 gi|4099506 gi|20070125

P30048 Q9NSD9 P18669 P07737 O95831 Q9UQ80 P07237

21158 66757 28916 15068 66539 38321 57480

6.1 6.4 6.7 8.4 9.0 7.2 4.8

117 187 102 216 180 225 360

38 20 30 44 24 17 27

oxidoreductase biosynthesis hydrolase actin binding apoptosis signal transduction chaperone

228 183

1 1

protein disulfide isomerase-related protein 5 gi|1710248 protein disulfide-isomerase A3 gi|860986

Q14554 P30101

46512 57043

5.0 6.1

676 439

57 44

oxidoreductase isomerase

343

1

gi|4506005

P62140

37961

5.8

235

20

phosohatase

272 173 83 82 302 259 99 342

1 1 1 1 2 1 1 1

gi|215261382 gi|67464392 gi|22477159 gi|13529299 gi|157835884 gi|78057661 gi|14530105 gi|4506003

P08559 P14618 Q15436 Q15437 Q15019 Q8TC62 Q9UHD8 P62136

43164 60277 87022 87377 36418 48913 64894 38229

6.9 8.2 6.6 6.4 6.3 8.8 7.2 5.9

293 485 401 287 505 494 329 312

25 42 27 20 51 34 31 37

oxidoreductase glycolysis transport transport cell division cell division cell division multifunctional

SY,MT ME,EX,SY ME,ER,PL EN ME,EX,SY ME,PL,SY ME EX

615 194

1 1

gi|189017 gi|14043738

Q15746 Q9Y512

11753 52270

4.5 6.6

103 350

30 51

cell motility transport

ME ME,EX,MT

305 540 229

1 1 1

protein phosphatase 1, catalytic subunit, beta isoform 1 pyruvate dehydrogenase E1 pyruvate kinase SEC23A protein SEC23B protein septin 2 septin 7 septin 9 serine/threonine-protein phosphatase PP1-alpha catalytic subunit smooth muscle myosin light chain sorting and assembly machinery component 50 homologue stomatin-like protein 2 superoxide dismutase [Cu-Zn] talin 1

ME,EX,MT ER,ME LG ER,LY,EN,ME, NG,PL,MT ER,EN,ME, SY,MT ME,PL,MT EN,ME ME,EX,SY ME,EX,PL,MT MT,ER,ME ME,EN,ER ER,ME,EX, PL,MT ME,ER,PL ER,LY,ME, NG,EX,PL ME,PL

gi|2984585 gi|31615966 gi|55859707

Q9UJZ1 38839 P00441 16001 Q5TCU6 260030

6.4 5.7 6.1

418 112 163

49 45 4

ME,MT ME,EX,MT EN,ME,PL

158 153 385 254 132 539

1 1 1 1 1 2

gi|36796 gi|119628379 gi|18088455 gi|193885198 gi|388891 gi|5454090

P17987 P40227 Q15006 Q9BS26 P29401 P51571

60869 39434 34955 44284 68528 19158

6.0 6.7 6.2 5.0 7.9 5.8

159 300 115 357 486 190

19 36 31 31 34 38

467 191 178 389

1 1 1 1

T-complex protein 1 subunit alpha T-complex protein 1 subunit zeta tetratricopeptide repeat protein 35 thioredoxin domain-containing protein 4 transketolase translocon-associated protein subunit delta triosephosphate isomerase 1 tubulin beta vimentin voltage-dependent anion channel 1

receptor binding oxidoreductase cytoskeleton anchoring chaperone chaperone unknown chaperone ligase protein transport

gi|17389815 gi|18088719 gi|47115317 gi|4507879

P60174 P07437 P08670 P21796

26910 50096 53604 30868

6.5 4.8 5.1 8.6

87 467 770 794

18 51 70 80

biosynthesis microtuble movement movement channel

434

1

gi|25188179

Q9Y277

30981

8.9

296

49

channel

390

1

gi|48146045

P45880

30849

6.8

320

46

channel

SY,MT

199 201 332 405 402

1 1 1 1 1

gi|13938355 gi|522193 gi|542837 gi|87159816 gi|119574080

P21281 P21281 P61421 A8MUE4 P63244

55708 56792 32083 23630 30942

5.4 5.6 5.2 9.0 7.0

295 479 140 117 283

31 52 29 26 33

ion transport ion transport ion transport ion transport signal transduction

LY,ME,NG,SY LY,ME,NG,SY LY,EN,ME,SY LY ER

384 335

1 1

gi|24307901 gi|219842325

P80217 Q15555

31896 32444

5.8 5.3

227 106

25 27

immunity signal transduction

PL PL

198

1

voltage-dependent anion channel 3 isoform b voltage-dependent anion-selective channel protein 2 V-type proton ATPase subunit B2 V-type proton ATPase subunit B2 V-type proton ATPase subunit D1 V-type proton ATPase subunit E1 guanine nucleotide-binding protein subunit beta-2-like 1 interferon-induced protein 35 microtubule-associated protein RP/EB family member 2 tubulin alpha

ME,EX,SY,MT ME,PL,SY ME ER,LY,ME,NG, EX,PL,SY,MT ME,EN,MT

gi|340021

P68363

50804

4.9

524

46

PL

506

1

actin beta variant

gi|62897625

Q53G99

42080

5.4

128

13

230 124 122

1 1 1

Enah/Vasp-like L-plastin L-plastin variant

gi|7706687 gi|167614506 gi|62898171

Q9UI08 P13796 Q53FI1

44878 70814 70785

8.9 5.3 5.2

231 373 159

31 26 15

microtuble movement cytoskeleton movement actin binding calcium binding ion binding

1609

ER,EN,ME,EX ME,EX,PL ME,ER,NU ER,ME,PL EN,ME ER,ME

CY CY CY CY



ARTICLE

Table 1. Continued spot no. replicates 301

1

148 240

1 1

143

1

97 31 115

1 1 1

77 321 291 464 573 571 189

1 1 1 1 1 1 1

130 416 121

2 1 1

331

1

474 418

1 1

295 294

1 1

328

1

151

1

86 462

1 1

219 250 287 592 458

1 1 1 1 1

412

1

225 159 408

1 1 1

155

1

454 460 493 164 37 290

1 1 1 1 1 1

461 177

1 1

299 145

1 1

330

1

protein name

acc. no.

Uni-Prot no.

MW, Da

pI



function

serine (or cysteine) proteinase inhibitor, clade B, member 1 T-complex protein 1 subunit gamma tubulin gamma 1

gi|13489087

P30740

42857

5.9

531

34

inhibitor

CY

gi|671527 gi|31543831

P49368 P23258

60862 51480

6.2 5.8

203 291

21 31

CY CY

tyrosine-protein phosphatase nonreceptor type 6 dead box, X isoform DNA damage-binding protein 1 ubiquitin associated and SH3 domain-containing protein A ATP-dependent DNA helicase 2 budding uninhibited by benzimidazoles 3 flap endonuclease 1 high-mobility group box 2 histone cluster 1, H2bm histone H2B type 2-E PRP19/PSO4 pre-mRNA processing factor 19 homologue replication protein A1, 70 kDa replication protein A2, 32 kDa alkyldihydroxyacetone phosphate synthase precursor 28S ribosomal protein S22, mitochondrial 3-hydroxyacyl-CoA dehydrogenase type-2 3-hydroxybutyrate dehydrogenase, type 1 [Homo sapiens] 3-ketoacyl-CoA thiolase, mitochondrial acyl-CoA dehydrogenase, mitochondrial, medium chain acyl-CoA dehydrogenase, mitochondrial, short chain acyl-CoA dehydrogenase, mitochondrial, very long chain acyl-CoA synthetase adenylate kinase isoenzyme 4, mitochondrial ATP synthase subunit beta coenzyme Q6 homologue isoform b CS protein [Homo sapiens] cytochrome c oxidase subunit Vb cytochrome oxidase deficient homologue 2 delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial dihydrolipoamide succinyltransferase DnaJ homologue subfamily C member 11 electron transfer flavoprotein, alpha polypeptide electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial enoyl-CoA hydratase ETHE1 protein, mitochondrial glutathione S-transferase kappa 1 heat shock 60 kDa protein 1 variant 1 hexokinase-1 isocitrate dehydrogenase [NADP], mitochondrial metaxin-2 methylcrotonoyl-CoA carboxylase beta chain, mitochondrial mitochondrial ribosomal protein L38 NAD-dependent malic enzyme, mitochondrial NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42 kDa precursor [Homo sapiens]

gi|82407989

P29350

60638

6.1

128

12

chaperone microtuble movement hydrolase

gi|2580550 gi|2632123 gi|49640014

O00571 Q16531 P57075

73625 128086 70943

6.7 5.1 6.6

85 90 188

13 12 29

hydrolase protein binding degradation

CY,NU CY,NU CY,NU

gi|10863945 gi|4757880 gi|61680058 gi|11321591 gi|45767717 gi|184086 gi|7657381

P13010 O43684 P39748 P26583 Q99879 Q16778 Q9UMS4

83222 5.6 37587 6.4 42777 8.8 24190 7.6 14080 10.4 11324 10.1 55603 6.1

426 241 166 214 70 77 253

40 38 13 48 50 57 25

DNA binding protein binding hydrolase DNA binding DNA binding DNA binding DNA binding

NU NU NU NU NU NU NU

gi|4506583 gi|4506585 gi|4501993

P27694 P15927 O00116

68723 29342 73664

6.9 5.8 7.0

363 162 302

33 12 25

DNA binding DNA binding biosynthesis

NU NU PE

gi|119599451

P82650

36840

6.3

236

41

protein binding

MT

gi|55670219 gi|15080429

Q99714 Q02338

27130 38516

8.5 9.1

144 182

41 31

oxidoreductase oxidoreductase

MT ER,MT

gi|509676 gi|4557231

P42765 P11310

42469 47015

8.5 8.6

426 286

39 26

metabolism metabolism

MT,ER MT

gi|4557233

P16219

44611

8.1

106

13

metabolism

MT

gi|119610656

B3KPA6

62941

6.8

374

33

metabolism

MT

gi|4758332 gi|83754030

O60488 P27144

75471 28030

8.3 7.3

194 127

21 26

metabolism Kinase

MT,PE,ME EN,MT

gi|89574029 gi|32967307 gi|48257138 gi|119622335 gi|153791313

Q0QEN7 Q9Y2Z9 O75390 P10606 O43819

48083 5.0 43389 6.9 45758 6.5 19755 10.0 29962 9.0

688 130 434 130 106

69 18 28 28 19

ion transport oxidoreductase transferase electron transport redox protein

MT MT MT,PL ER,MT MT

gi|183448176

Q13011

33061

6.4

474

38

isomerase

MT

gi|736677 gi|37732147 gi|189181759

P36957 Q9NVH1 Q53XN3

48992 59138 30235

9.0 7.2 8.8

284 88 76

33 14 13

transferase chaperone electron transport

MT MT MT

gi|31874555

Q16134

65502

6.9

380

24

electron transport

MT

gi|1922287 gi|41327741 gi|33150564 gi|189502784 gi|188497750 gi|119622488

P30084 O95571 Q9Y2Q3 B3GQS7 P19367 P48735

31807 28368 25580 60813 102161 48071

8.3 6.4 8.5 5.8 6.5 8.3

281 124 192 380 147 546

20 25 35 42 11 57

lyase hydrolase transferase chaperone glycolysis metabolism

MT MT,CY,NU MT,ME,PL MT PL,MT PL,MT

gi|62702262 gi|11545863

O75431 Q9HCC0

28668 61808

6.1 7.6

67 290

12 40

protein transport ligase

MT MT

gi|169636418 gi|31615313

Q96DV4 P23368

44968 62563

7.2 6.4

364 170

45 14

unknown oxidoreductase

MT PL,MT

gi|4758768

O95299

41067

8.7

405

45

electron transport

MT

1610

CY



ARTICLE

Table 1. Continued spot no. replicates 574

1

232

1

278

1

46

1

577

1

277

1

138

2

293

1

262

1

388 111

1 1

18 583 234

1 1 1

226

1

235 341

1 1

414

1

107

1

125 339

1 1

69 110 491

1 1 1

312 526

1 1

93 484 188 424 508 104 212 432 196

1 1 1 1 1 1 1 1 1

334 131 109

1 1 1

protein name NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial NADH dehydrogenase-ubiquinone Fe-S protein 2 precursor [Homo sapiens] oxoglutarate (alpha-ketoglutarate) dehydrogenase isoform 1 single-stranded DNA-binding protein, mitochondrial solute carrier family 25 member 24 isoform 1 succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial sulfide:quinone oxidoreductase, mitochondrial thiosulfate sulfurtransferase trifunctional enzyme subunit alpha, mitochondrial 150 kDa oxygen-regulated protein ACTG1 protein ARP3 actin-related protein 3 homologue variant calcium/calmodulin-dependent protein kinase type II delta chain DNA FLJ78579, highly similar to CSK erythrocyte 26 S protease subunit 12, 26 S hCG2016179, isoform CRA_d [Homo sapiens] heat shock 70 kDa protein 8 isoform 2 variant hypothetical protein SBBI88 isocitrate dehydrogenase 3 (NADþ) alpha KLRAQ motif containing 1 isoform 3 KU antigen loss of heterozygosity 12 chromosomal region 1 protein migration-inducing gene 14 myosin regulatory light chain MRCL3 variant NADH-ubiquinone oxidoreductase NDUFV2 protein OXCT protein PHB protein phorbolin I protein protein disulfide isomerase septin 6 SH3-domain binding protein 1 tubulin alpha 6 variant unnamed protein product unnamed protein product X-ray repair complementing defective repair in Chinese hamster cells 6 (Ku autoantigen, 70 kDa)

Uni-Prot no.

MW, Da

pI

gi|4680717

Q9P0J0

15729

6.9

148

34

electron transport

MT

gi|119595060

P49821

50707

8.5

257

33

electron transport

MT

gi|3540239

O75306

52869

7.2

327

39

electron transport

MT

gi|51873036

Q02218

117059

6.4

425

25

glycolysis

MT

gi|2624694

Q04837

15186

8.2

264

65

DNA binding

MT,PL

gi|148491091

Q6NUK1

53548

6.0

385

41

ion transport

MT

gi|156416003

P31040

73672

7.1

533

45

oxidoreducatse

ER,MT

gi|55957259

Q9P2R7

48351

6.6

189

14

protein binding

MT

gi|10864011

Q9Y6N5

50214

9.2

579

56

oxidoreductase

MT

gi|48146113 gi|20127408

Q16762 P40939

33622 83688

6.8 9.2

119 434

29 35

transferase oxidoreductase

PL,MT PL,MT

gi|193785329 gi|40225338 gi|62088286

B3KXH0 Q96FU6 Q59FV6

110601 18724 42517

5.2 5.2 5.6

350 241 258

15 38 31

unknown unknown unknown

gi|119626702

Q13557

59062

7.0

243

15

ATP binding trafficking cytoskeleton movement kinase

gi|158254494 gi|998688

A8K3B6 unknown

51240 36674

6.6 6.2

284 398

27 47

protein binding degradation

unknown unknown

gi|119617243

A5YM67

21868

5.9

132

27

chaperone

unknown

gi|62896815

Q53HF2

53580

5.6

150

22

chaperone

unknown

gi|6942315 gi|62897507

Q9Y3Z3 Q53GF8

72839 40050

6.6 6.5

366 173

36 11

hydrolase oxidoreductase

unknown unknown

gi|208431757 gi|189055002 gi|119616665

Q6ZMI0 B2RDN9 Q969J3

83659 70026 13442

6.2 6.3 6.3

109 101 84

12 9 48

unknown DNA binding unknown


gi|38570361 gi|62896697

Q5J8M4 Q53HL1

40080 19838

8.0 4.7

72 113

19 36

unknown cell motility

unknown unknown

gi|194373941 gi|48145973 gi|48146215 gi|49456373 gi|9294747 gi|158255378 gi|1469179 gi|85815829 gi|62897609

B4DUC1 Q6IB76 Q6IAV5 Q6FHP5 Q9NRW3 A8K4K6 Q14141 Q9Y3L3 Q53GA7

74393 27617 56612 29871 34310 73157 48911 76008 50476

5.6 8.2 7.1 5.6 5.5 5.0 6.4 6.3 5.0

286 198 128 314 164 163 340 156 107

37 48 14 50 19 17 44 6 17

unknown unknown unknown unknown unknown unknown unknown unknown unknown

gi|158256528 gi|194382854 gi|169145198

A8K683 B4DJX1 B1AHC7

35187 63057 64263

5.2 5.5 6.4

73 222 510

14 24 45

unknown oxidoreductase unknown unknown hydrolase isomerase cell division transport microtuble movement unknown protein binding unknown

acc. no.



function

unknown


a

A total of 240 individual proteins of 379 identified spots (see Supplemental Table S1, Supporting Information, and individual data sets) are listed. Proteins are alphabetically sorted according to their subcellular localization, starting with lysosomal-related organelles. The table lists the spot number with the number of iterant identifications, the respective accession number of the proteins in NCBI and Uni-Prot, the theoretical molecular weight (MW) and isoelectric point (pI). The resulting total MASCOT score and sequence coverage as well as the assigned protein function, and subcellular localization of the respective protein are given. Abbreviations: LY, lysosomes; ME, melanosomes; PL, platelet granules; SY, synaptosomes; EX, exosomes; CG, cytotoxic granules; NG, neuromelanin granules; EN, endosomes; MT, mitochondria; GO, Golgi; PE, peroxisomes; CY, cytoplasm; ER, endoplasmic reticulum; NU, nucleus. 1611



ARTICLE

and 23 spots were more abundant in the fraction 6 samples with a t test value of e0.05. Importantly, 105 of the 120 differential spots were identified based on the previously established spot maps (Table 3). Verification of Fraction-Specific Proteins

Figure 5. Subcellular distribution of the identified proteins. A total of 240 individual proteins were identified and grouped according to their subcellular localization based on available database entries. Abbreviations are used as in Table 1.

with lysosomal or secretory compartments based on previous studies.14 This percentage is in the same order of magnitude as the numbers we reported for fraction 2 vesicles from NK cells or T cell blasts and indicates that fraction 2 and fraction 6 vesicles might indeed represent two separate lysosomal compartments. Notably, in fraction 6 vesicles, we found significant amounts of granzymes A and B, perforin and several lysosomal hydrolases including the cathepsins B, D, S and W. The proteome map of fraction 6 organelles is depicted in Figure 4. For a better visualization of individual annotated spots, enlarged quadrants are displayed in supplemental Figure S6A-D (Supporting Information). A short list of identified proteins, ordered alphabetically according to their predicted organelle localization is given in Table 1. In addition, the predicted organelle association is summarized as a pie chart in Figure 5. The complete list of proteins including relevant data of the mass spectrometric identification is provided as Supplemental Table S1 (Supporting Information). Individual data sets for each spot are also given in the Supporting Information. Direct Comparison of Fraction 2 and Fraction 6 Vesicles

To directly compare the protein content of the two putative effector organelles, we subjected equal amounts of the two fractions obtained from PHA-stimulated T lymphocytes of four different donors to 2D-DIGE analyses (Table 2). As an example, one biological replicate is displayed in Figure 6 where fraction 2 proteins were labeled with Cy5 and fraction 6 proteins with Cy3. From the overlay picture (Figure 6A) and also from the representation of the individual scans (Figure 6B,C), major differences in the overall spot distribution became immediately apparent. This was seen in all biological replicates (Figure S2, Supporting Information). The software-based analysis of the in-gel differences (DIA) resulted in a calculated 2-fold square deviation of 1.61 when comparing fraction 2 samples from two different donors (Figure 7A). In contrast, applying the same calculation for the comparison of fraction 2 and 6 from the same donor, the 2-fold square deviation was 6.59 (Figure 7B). Accordingly, setting the threshold of the volume ratio to 6 resulted in a similarity of 100% comparing individual fraction 2 lysates whereas only 78.9% of the included spots (=proteins) were comparably abundant in fractions 2 and 6. These major differences in the overall protein composition were confirmed by the biological variance analysis listed in Table 2. Considering donor and gel specific variations, the number of spots included in the analysis was reduced to 801. Applying a threshold of 5.5, 97 of 801 spots were underrepresented

Selected examples of the differentially distributed proteins are shown in Figure 8. Some proteins were present in only one of the two compared fractions. These included β-actin, dipeptidylpeptidase IV (DPP4), moesin and cathepsin D in fraction 2, and granzymes A and B, myosin IIA and dynamin II in fraction 6. In addition to the proteins listed in Table 3, we verified further differences in potentially functionally relevant proteins that were not included in the table because of a high t test value or a lower volume ratio (Figure S3, Supporting Information). Among them are Nck and WASp, two proteins enriched in fraction 2 (Figure S4A, Supporting Information) and involved in the transport of FasL.13,19 Regarding fraction 6, we confirmed our previous Western blot analyses10 indicating that granzyme A and granzyme B were much more abundant in these vesicles (see 3D representation in Figure 8C). Note that we did not include granzymes A and B in Table 3 because of high t test values due to differences in the resolution in the basic parts of the IPG strips. However, also the direct comparison of the two fractions from T cell blasts of two individuals by Western blot analysis clearly showed the differences between fraction 2 and 6 (Figure 8D). Since flotilin-1 spot volumes were comparable between the fractions, it was used as an internal loading control (Figure S3C, Supporting Information). More examples for differentially abundant proteins are displayed in supplemental Figure S3A,B, Supporting Information. Interestingly, major differences were especially noted for cytoskeletal or cytoskeleton-regulating proteins. Thus, β-actin, actinin alpha 4 and coronin-1A were more abundant in fraction 2 isolates, whereas γ-actin, myosin light chain kinase and myosin IIA (myosin 9) were associated with fraction 6. For myosin IIA, a detailed description of the biological variance analysis is given in the Supporting Information, once more stressing the high reproducibility of the performed organelle fractionation and protein separation when comparing samples from different donors (Figure S5, Supporting Information).

’ DISCUSSION The maturation-dependent storage of FasL as a transmembrane component of secretory lysosomes is a commonly accepted feature of fully differentiated cytotoxic immune effector cells. Upon exposure to target cells, the availability of prestored FasL allows its rapid surface appearance at the immunological synapse. This is reflected by a biphasic kinetics of activationinduced FasL expression, with an actin-dependent phase 1 expression within minutes and a protein-biosynthesis-dependent phase 2 expression after hours.5,6 However, effective target cell lysis does not only rely on the membrane appearance of FasL, but rather depends on a concerted action of FasL with a number of secreted proteins including granzymes, perforin and granulysin. Interestingly, although earlier studies suggested a common lysosomal storage compartment for all effector proteins,3,4,18,20 more detailed analyses on the signal requirements for mobilization of effector proteins recently indicated the coexistence of separate compartments for FasL and factors associated with degranulation.7,8 1612



ARTICLE

Table 2. Overview of DIGE Experiments Performed to Directly Compare Fraction (F) 2 and 6 Vesicles from T Lymphoblasts of Four Different Donorsa included spots gel #

Cy5 labeling

Cy3 labeling

#

increased in fraction 6 2 S.D.

#

%

decreased in fraction 6 #

%

1

donor I F2

donor I F6

1340

6.9

255

19.0

59

4.4

2

donor II F2

donor II F6

1122

5.2

42

3.7

110

9.8

3 4

donor III F6 donor IV F6

donor III F2 donor IV F2

1487 1207

3.9 6.6

36 129

2.4 10.7

56 125

3.8 10.4

average:

1289

5.6

116

9.0

88

6.8

included in BVAb:

801

23

97

a

The 2-fold square deviation (2 S.D.) indicates the difference between the compared fractions. The numbers of increased and decreased spots refer to a spot volume threshold of 5.5. b Biological variance analysis (BVA) was performed with all gels, only spot present in at least three donors were included. The number of regulated spots is based on a volume ratio change of 5.5 and a t test of e0.05.

Figure 6. 2D-DIGE analysis of fraction 2 and 6 isolates. Proteins of fraction 2 and 6 organelles were labeled with Cy5 (red) and Cy3 (green), respectively. The samples were separated by two-dimensional electrophoresis as described in the Material and Methods section. Images of individual fluorescence channels are displayed for fraction 2 in (B) and for fraction 6 in (C). Spots with higher abundance in fraction 6 are circled in blue and with lower abundance circled in red.

At the resolution of microscopic inspection and confocal imaging, we and others reported a colocalization of FasL with lysosomal markers such as CD63, LAMP-1 (CD107), cathepsin D and also with granzymes and perforin.3,4,13,18 However, this has been challenged by the work of He et al.6 and Kassahn and colleagues.8 Using a murine CD8þ T cell clone and employing similar hardware but additional deconvolution software and 3D imaging to presumably render more precise images, He and

colleagues in fact suggested the existence of two types of storage granules for FasL and for granzymes, respectively.6 Nevertheless, although deconvolution might slightly enhance the resolution compared to standard confocal laser scanning microscopy, most previous reports by Griffiths and colleagues1-4 and our own observations suggested to a colocalization or at least a very close proximity of the effector compartments in most analyzed T and NK cell populations. Since it seems unlikely that distinct granules 1613



ARTICLE

Figure 7. Statistical representation of the overall protein distribution (A) in fraction 2 extracts of T cell blasts from two donors and (B) in fraction 2 and 6 extracts from one individual. The red line indicates the number of spots with a specific abundance ratio (log scale). The blue line represents the calculated Gaussian distribution. Red dots represent decreased and blue increased protein volumes in individual spot locations of fraction 6. The threshold in both analyses was set to 5.5 (vertical lines).

occupy closely adjacent cytosolic areas by chance, a more sound explanation for the apparent discrepancy might be that the differentially loaded effector granules are at least partially stored in a common multivesicular compartment. However, we have to admit that we so far failed to convincingly document this multivesicular compartment in fixed T cell blasts by electron microscopy using a set of presumably granule-specific antibodies and immunogold labeling. Another caveat to the interpretation of confocal images regarding colocalization might be that the cells/ areas were somehow “selected for colocalization”. In our own studies, we noted that protein distribution and localization is not homogeneous in all cells of a given (even clonal) population, with some cells displaying a more intense FasL and others a brighter granzyme/perforin staining. Thus, the composition of the effector vesicle compartment might be subject to change due to activation or nutritional parameters. A coexistence of two separate effector compartments becomes very likely considering the data obtained in the present study. After density gradient centrifugation and Western blot analysis, the marker proteins clearly segregate in different fractions. While FasL and CD63 are enriched in fraction 2, granzyme B and the active form of granulysin are present in fraction 6 (Figure 2). As visualized by electron microscopy, vesicles in individual fractions are quite homogeneous. However, fraction 2 vesicles are mostly larger and less electron dense than fraction 6 vesicles. Notably, organelles resembling multivesicular bodies were only seen in fraction 2 (Figure 3A, S1A). Interestingly, only fraction 6 vesicles contained a less electron dense structure that suggested polarity and therefore was reminiscent of a

earlier description of an electron-translucent space of unknown nature in cytolytic granules of decidual γδ T cells.21 The localization of granzymes A and B in electron dense structures could be in agreement with their reported tight packaging in multimolecular complexes associated with chondroitin-sulfate proteoglycans of the serglycin type.22 In this context, it was shown earlier that such complexes are delivered by exocytosis to induce apoptosis in target cells.23 Granulysin is a cationic member of the family of saposin-like proteins (SAPLIPs) and as such interacts with lipids.24 Western blot analyses revealed that granulysin is detected as a 15 kDa form in fraction 2 vesicles, whereas a 9 kDa form is seen on the same blot in fraction 6. The processing of the proform to the active smaller form25 requires posttranslational modifications of both the N-terminus and the C-terminus in an acidic environment26 and only the active 9 kDa form is able to lyse bacteria and fungi.27,28 It has been reported that in certain situations, the lytic activity of granulysin can be enhanced by perforin.29 In this context, it makes sense that also perforin was present in significantly higher amounts in fraction 6 vesicles (Figure S4C, Supporting Information). For the detection of perforin, we used a recently established antibody (clone B-D48) which supposedly reacts with multiple forms of the protein while the classical mAb (clone deltaG9) only recognizes the granule-associated conformation.30 Interestingly, higher molecular weight bands at approximately 65 and 70 kDa were only detected in fraction 2, whereas high amounts of the presumably C-terminally processed 60 kDa form of perforin were predominant in fraction 6.31 1614



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Table 3. List of Differentially Abundant Protein Spots in Fraction 2 and 6 Vesiclesa BVA spot

protein ID

protein ID

no.

fraction 2

fraction 6

average volume protein name

acc. no.

t test value

ratio

1406

841

coactosin-like protein

gi|56966036

6.3 10-5

-22.2

1384

820

stathmin 1/oncoprotein 18

gi|122890670

2.2 10-4

-21.4

1271

728

Rho GDP dissociation inhibitor (GDI) beta

gi|56676393

2.1 10-5

-21.0

1465

nd

7.2 10-5

-20.9

241

143

actinin; alpha 4

gi|12025678

1.2 10-6

-18.4

953

522

ACTB protein

gi|15277503

7.6 10-8

-17.4

39 966

15 508

coronin; actin binding protein; 1A ACTB protein

gi|5902134 gi|15277503

5.4 10-4 5.5 10-8

-17.4 -17.2

633

371

coronin; actin binding protein; 1A

gi|5902134

6.3 10-6

-17.1

936

508

ACTB Protein

gi|15277503

2.2 10-5

-16.9

890

485

ACTB protein

gi|15277503

3.3 10-3

-16.3

940

522

ACTB protein

gi|15277503

1.3 10-7

-16.2

935

525

ACTB protein

gi|15277503

2.0 10-2

-15.8

955

508

ACTB protein

gi|15277503

3.7 10-7

-15.6

1251 508

nd 266

not identified L-plastin

gi|4504965

5.4 10-5 2.1 10-5

-15.3 -14.6

238

140

actinin; alpha 4

gi|12025678

2.0 10-7

-14.4

939

526

ACTB protein

gi|15277503

3.0 10-6

-14.1

1265

nd

not identified

1.2 10-3

-14.1

1279

742

triosephosphate isomerase 1

gi|4507645

4.5 10-6

-14.1

1178

676

tropomyosin 3 isoform 2

gi|24119203

1.8 10-5

-13.8

636

372


gi|5902134

9.2 10-6

-13.3

497 931

267 527

L-plastin

variant ACTB protein

gi|62898171 gi|15277503

6.7 10-6 5.9 10-5

-13.3 -13.0

937

522

ACTB protein

gi|15277503

6.2 10-3

-12.8

686

397

adenylyl cyclase-associated protein variant

gi|62896585

6.3 10-5

-12.6

509

275

L-plastin

gi|4504965

3.0 10-2

-12.4

1379

810

cofilin 1

gi|5031635

3.0 10-3

-12.3

942

525

ACTB protein

gi|15277503

5.3 10-6

-11.7

516

299

WD repeat-containing protein 1 isoform 1 variant

gi|62897087

1.4 10-4

-11.2

506 34

275 8

L-plastin


gi|4504965 gi|1002923

7.8 10-3 9.8 10-6

-11.0 -10.8

684

404


gi|62896585

2.3 10-5

-10.4

803

960

lymphocyte-specific protein 1

gi|10880979

2.2 10-4

-10.4

121

49


gi|1002923

1.6 10-7

-10.4

1380

814

destrin isoform a

gi|5802966

2.0 10-2

-10.3

816

460

solute carrier family 9 (sodium/hydrogen

gi|4759140

6.2 10-6

-9.8

8.8 10-5 6.0 10-4

-9.7 -9.6

not identified

exchanger); isoform 3 regulator 1 499 988

269 568

gelsolin-like capping protein isoform 9

gi||4504965 gi|55597035

1193

684

nuclear chloride channel

gi|4588526

7.7 10-4

-9.5

242

144

actinin; alpha 4

gi|119577215

8.2 10-5

-9.4

442

263

annexin A6

gi|35218

1.8 10-2

-9.3

1100

642

cytosolic malate dehydrogenase

gi|5174539

1.5 10-4

-9.1

239

141

Rap1-GTP-interacting adapter molecule

gi|26000235

1.5 10-5

-9.0

1080

630

LIM and SH3 domain protein 1

gi|1584035

8.2 10-7

-9.0

595 927

351 524

WD repeat domain 1 ACTB protein

gi|119613095 gi|15277503

1.6 10-6 3.0 10-5

-8.9 -8.7

695

395


gi|62896585

2.3 10-5

-8.6

517

303

WD repeat-containing protein 1 isoform 1

gi|9257257

7.3 10-3

-8.6

1256

716

Rho GDP dissociation inhibitor (GDI) alpha

gi|4757768

1.0 10-3

-8.6

1420

847

profilin-1

gi|5822002

4.3 10-6

-8.4

1151

660

annexin A5

gi|809185

2.5 10-6

-8.4

L-plastin

1615



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Table 3. Continued BVA spot

protein ID

protein ID

no.

fraction 2

fraction 6


acc. no.

t test value -5

ratio

1098

641

G protein beta subunit

gi|306785

8.7 10

-8.3

192

112

heat shock protein 70

gi|292160

1.5 10-2

-8.0

1249

730

PGAM1

gi|49456447

1.9 10-4

-7.9

857

496

enolase 1 variant

gi|62896593

7.3 10-4

-7.9

615

360


gi|5902134

2.0 10-6

-7.8

496

268

L-plastin

gi|4504965

1.4 10-4

-7.8

1348

787

transgelin-2

gi|9956026

2.6 10-3

-7.8

237 1316

1052 766

actinin; alpha 4 glutathione S-transferase P1

gi|119577215 gi|20664358

1.7 10-3 1.3 10-3

-7.7 -7.7

1385

818

glia maturation factor gamma

gi|19697925

5.3 10-4

-7.5

1221

703

proteasome activator complex subunit 1 isoform 1

gi|5453990

7.3 10-4

-7.4

515

298

WD repeat-containing protein 1 isoform 1 variant

gi|62897087

2.9 10-4

-7.3

957

537

phosphoglycerate kinase 1

gi|48145549

4.0 10-4

-7.2

1417

848

profilin-1

gi|5822002

1.7 10-4

-7.1

390

237

moesin; isoform CRA_b

gi|119625804

1.3 10-3

-7.1

1070 934

628 281

annexin A2 ACTB protein

gi|56966699 gi|15277503

4.7 10-6 2.7 10-5

-7.0 -7.0

1107

649


gi|306785

6.8 10-4

-6.7

1176

672

tropomyosin 4

gi|4507651

1.4 10-3

-6.9

566

nd

not identified

4.4 10-4

-6.8

504

nd

not identified

3.2 10-2

-6.7

352

208

ezrin

gi|46249758

4.6 10-4

-6.4

1114

646


gi|306785

2.1 10-5

-6.4

1001 263

574 154

twinfilin-like protein ubiquitin specific peptidase 5 isoform 2

gi|6005846 gi|148727247

1.5 10-4 2.1 10-4

-6.3 -6.3

696

346

pyruvate kinase 3 isoform 2

gi|67464392

5.7 10-4

-6.3

1042

604

annexin A1

gi|4502101

1.2 10-5

-6.3

1062

622

calcium binding protein 39

gi|7706481

2.1 10-4

-6.3

963

548

serpin peptidase inhibitor; clade B; member 1

gi|62898301

7.7 10-4

-6.1

705

407

tubulin; beta

gi|18088719

5.8 10-4

-6.1

929

533/1016

MHC class I antigen

gi|64976582

5.7 10-4

-6.1

195 197

113 nd

coronin 7 not identified

gi|109658548

2.5 10-4 4.4 10-3

-6.1 -6.1

962

550

adenosine deaminase

gi|1197210

8.9 10-5

-6.1

1236

714

14-3-3 protein beta

gi|4507949

2.3 10-3

-6.0

1057

615

annexin A1

gi|4502101

1.6 10-6

-6.0

1149

659

annexin A5

gi|809185

1.7 10-3

-6.0

363

209

interleukin-16

gi|27262655

1.1 10-3

-5.9

1368

nd

not identified

6.6 10-2

-5.9

699 1071

390 629

phosphoglucose isomerase gamma-glutamyl hydrolase

gi|14488680 gi|4503987

8.2 10-4 1.8 10-3

-5.9 -5.9

493

269

L-plastin

gi|4504965

3.0 10-3

-5.8

821

469

Rab GDP dissociation inhibitor beta

gi|119606836

6.9 10-6

-5.7

1376

812

cofilin 1

gi|5031635

1.9 10-3

-5.7

1453

865

MHC class I antigen

gi|229995

2.1 10-4

-5.6

myosin 9

gi|12667788

5.7 10-3

5.5

243

270

63

1202

429

PHB protein

gi|49456373

2.3 10-2

5.5

382 1361

95 526

NADH-ubiquinone oxidoreductase 75 kDa subunit myosin regulatory light chain MRCL3 variant

gi|21411235 gi|62896697

8.1 10-3 4.4 10-2

5.5 5.6

594

nd

4.0 10-2

5.7

1199

434

voltage-dependent anion channel 3 isoform b

gi|25188179

7.6 10-3

5.8

912

295

3-ketoacyl-CoA thiolase; mitochondrial

gi|509676

7.7 10-3

6.0

924

287

CS protein [Homo sapiens]

gi|48257138

1.2 10-2

6.0

not identified

1616



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Table 3. Continued BVA spot

protein ID

protein ID

no.

fraction 2

fraction 6


acc. no.

t test value -3

ratio

1275

nd

not identified

1.2 10

6.0

1255

nd

not identified

1.5 10-3

6.2

1145

nd

not identified

4.0 10-4

6.3

1415

583

ACTG1 protein

gi|40225338

1.1 10-3

6.6

1143

391

voltage-dependent anion channel 1

gi|4507879

4.0 10-3

6.7

164

chaperonin (HSP60)

gi|306890

4.7 10-2

6.8

1407

577

single-stranded DNA-binding protein; mitochondrial

gi|2624694

1.5 10-3

6.9

1393 1427

615 592

smooth muscle myosin light chain cytochrome c oxidase subunit Vb

gi|189017 gi|119622335

1.0 10-3 2.2 10-3

7.0 7.1

1428

594

ACTG1 protein

gi|40225338

1.1 10-3

7.3

1144

389

voltage-dependent anion channel 1

gi|4507879

1.2 10-3

7.4

1277

nd

not identified

1.3 10-3

7.7

764

nd

not identified

1.4 10-2

7.9

1186

416

267

63

601

338

replication protein A2; 32 kDa

gi|4506585

1.1 10-2

7.9

myosin 9

gi|12667788

2.5 10-3

7.9

a

Independent 2D-gels of fractions 2 and 6 samples obtained from lymphocytes of four donors were statistically evaluated using biological variance analysis (BVA). All included spots of fraction 2 and 6 samples are represented with their spot number in the corresponding mastergel. Parameters for variance analysis were set to p > 0.05 in t-test analysis and an average spot volume ratio >5.5 and 17). This is of special interest with regard to the ongoing controversy how transport of vesicles and degranulation are regulated by actin-associated elements. Actinin-alpha 4 (Figure S3A, Supporting Information), present in fraction 2, supports the bundling of actin filaments and thereby takes part in the regulation of actin-mediated exocytosis.41 Coronin controls actin dynamics through coordinated effects on the Arp2/3 complex and cofilin.42 By contrast, fraction 6 granules contained significantly higher amounts of myosin IIA (myosin 9), the functionally associated calcium/calmodulin-dependent myosin light chain kinase (MLCK), the myosin regulatory light chain 3 (MRLC3), and 1617



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Figure 8. Verification of the different abundance of protein spots in individual fractions. Selected proteins with a higher abundance in fraction 2 are displayed as 3D images in (A). The results were verified by Western blotting for two different donors with specific antibodies (B). In contrast, the proteins displayed in (C) were enriched in fraction 6. Respective Western blot verification is shown in (D). Numbers indicate the fold change in volume ratios. Flotilin-1 abundance was unchanged and therefore served as a loading control (WL = whole lysate).

γ-actin (factor >7). In fact, MLCK, a key regulator of myosin II that has been implicated in vesicle release at neuronal synapses,43 was only detectable by mass spectrometry in fraction 6 samples (Figure S3B, Supporting Information). It was suggested that these proteins selectively regulate degranulation. Thus, inhibition of myosin IIA by blebbistatin blocked granzyme B secretion in human NK cells while the polarization of the microtubule organizing center (MTOC), the formation of a characteristic actin-ring and the development of a functional immunological synapse were not affected.44 In support of the differential mobilization hypothesis, it was later shown that blebbistatin did not abolish TCR- or PMA-induced FasL killing.8 Also dynamin II was enriched in fraction 6 sample as evidenced by DIGE and Western blot analyses (Figures 8C,D and S4B, Supporting Information). It is well established that dynamin II is involved in the regulation of the final fusion of the lysosomal and the plasma membrane during the kiss of death.45 Our results thus provide a further explanation why granzyme A secretion is blocked upon dynamin II inhibition.46 With these results in mind, one may hypothesize that the two species of effector vesicles are differently connected to the

cytoskeleton. Recently, using a set of anti-actin antibodies that apparently do not crossreact to other actin forms, Dugina and coworkers compared the distribution of cytoplasmic β- and γ-actin in fibroblasts and epithelial cells.47 They observed that γ-actin was distributed into the cortical microfilamental meshwork at the lamellar periphery, while β-actin was localized in circular bundles and in radial stress fibers. Translating this to the situation in cytotoxic cells, one could speculate that electrondense, granzyme-rich fraction 6 vesicles might be associated with the γ-actin meshwork close to the plasma membrane, whereas secretory lysosomes (fraction 2) might be connected to the fibrilliar β-actin network and are transported to the immunological synapse in a myosin IIA -and microtubuleindependent manner.6,8 The possible distinction between a myosin-dependent degranulation and an β-actin-dependent FasL transport would at least in part explain the recent studies on FasL release. The biphasic surface appearance of FasL5,6 appears to coincide with different signaling requirements and thresholds.7,8 Moreover, it was shown that the rapid release of prestored FasL does not require Ca2þ, whereas the expression of newly synthesized FasL is Ca2þ 1618


Journal of Proteome Research dependent.8 It is well established that the concentration of available Ca2þ correlates with the strength of the TCR signal. Thus, FasL is rapidly released in response to weak TCR signals while de novo synthesized FasL and degranulation (of granzyme pools) require strong TCR signals.9,7,48 In this context, it might be of interest that a bimodal secretion of cell-cell-communication molecules has not only been described for polarized neuronal or epithelial cells but also for T helper cells.49 Huse and colleagues recently reported that T helper cells release for example IL-2 and interferon-γ into the immunological synapse, whereas TNF and the chemokine CCL3 (MIP-1R) are secreted multidirectionally. The two secretion pathways were associated with distinct trafficking proteins suggesting molecularly distinct processes. Thus, one might speculate that the different transport mechanisms could also be utilized for the mobilization of individual effector proteins. In essence, however, it seems common sense that at least in in vitro situations, FasL as well as perforin and granzymes work best in killing target cells when released into the cytotoxic immunological synapse. Nonetheless, further studies might reveal other more systemic functions of the individual molecules in cell-cell communication that are associated with distinct trafficking proteins and processes.

’ CONCLUSION With the present report, we provide for the first time strong biochemical/proteomic evidence for the coexistence of two distinct types of effector vesicles in activated human T cells. We describe the luminal proteome of granzyme-containing granules and directly compare these granules with FasL-associated secretory lysosomes by 2D-DIGE. Our observations support the hypothesis that mobilization of FasL-containing vesicles and the degranulation of granzyme-containing granules rely on distinct signals that also might involve different cytoskeletal elements. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Prof. Dr. Ottmar Janssen, PhD, Christian-Albrechts-University, Institute of Immunology, UK S-H Campus Kiel ArnoldHeller-Str. 3 Bldg. 17, D-24105 Kiel, Germany. Phone: þ49 431 5973377. Fax: þ49 431 5973335. E-mail: ojanssen@email. uni-kiel.de.

’ ACKNOWLEDGMENT This work was supported by grants from the German Research Foundation (SFB 415 and SFB 877), the Cluster of Excellence “Inflammation at Interfaces”, the Innovationsfond SchleswigHolstein, and the Medical Faculty of the Christian-AlbrechtsUniversity of Kiel. ’ REFERENCES (1) Stinchcombe, J.; Bossi, G.; Griffiths, G. M. Linking albinism and immunity: the secrets of secretory lysosomes. Science 2004, 305 (5680), 55–59.

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Effector Granules in Human T Lymphocytes - American Chemical

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