Interaction of Insulin-Like Growth Factor-Binding protein 3 With

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Interaction of Insulin-Like Growth Factor-Binding protein 3 With Hyaluronan and Its Regulation by Humanin and CD44 Robert Muterspaugh, Deanna Price, Daniel Esckilsen, Sydney McEachern, Jeffrey Guthrie, Deborah L. Heyl, and Hedeel Guy Evans Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00635 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Biochemistry

Interaction of Insulin-Like Growth Factor-Binding protein 3 With Hyaluronan and Its Regulation by Humanin and CD44. Robert Muterspaugh, Deanna Price, Daniel Esckilsen, Sydney McEachern, Jeffrey Guthrie, Deborah Heyl, and Hedeel Guy Evans* Chemistry Department, Eastern Michigan University, Ypsilanti, Michigan 48197, United States Corresponding Author *E-mail: [email protected]. Phone: (734) 487-1425. Fax: (734) 487-1496.

ABSTRACT: Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of IGF binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (215-KKGFYKKKQCRPSKGRKR232)

to HA and to humanin and found that HA binds with a weaker affinity to this region

than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in media of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the media that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it

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inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.

Table of Contents Graphic

CD44

HA

HA

HA

HA

CD44

IGFBP-3 Humanin

HA

HA

HA

HA HA

HA HA-CD44 Interaction Off

HA-CD44 Interaction On

IGFBP-3 binds to HA and blocks its interaction with CD44. Humanin counteracts this effect by binding to IGFBP-3 thus disabling its binding to HA which is now free to interact with CD44.

TOC Graphic fit to dimensions required by the journal.


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Biochemistry

INTRODUCTION Protein-peptide interactions are implicated in many human diseases and known to play a crucial role in the undertaking of numerous cellular processes. 1 In higher eukaryotes, peptides have been shown to mediate up to 40% of protein–protein interactions.2–4 Relatively little is known, however, about the role proteins and peptides play in regulating and fine-tuning carbohydrate signaling. IGFBP-3 includes a 27-residue signal peptide that is cleaved off in the 264-residue mature protein that is predominantly secreted. The protein is multifunctional with diverse roles in the circulation and in both the intra- and extra-cellular environment.5–7 IGFBP-3 belongs to a family of six IGF binding proteins that have highly conserved structures. Acting as a carrier and most abundant circulating IGFBP, IGFBP-3 regulates Insulin-like growth factor I (IGF-I) bioavailability and cell survival by IGF-dependent and independent mechanisms.8,9 IGFBP-3 can exert its antiproliferative functions by attenuating IGF/IGFIR interactions but also independently of the IGF/IGF-IR axis.5,10–12 Its expression is reduced13 in lung cancer which was found to be associated with poor diagnosis in stage I non-small-cell lung carcinoma (NSCLC) patients.14–18 An inverse relationship was demonstrated between serum or plasma levels of the protein and lung cancer risk. Noncytotoxic doses of IGFBP-3 were shown to significantly decrease the migration and invasion of A549 NSCLC cells.19 IGFBP-3 was found to suppress expression of urokinase-type plasminogen activator and matrix metalloproteinase-2, in part, through unidentified IGF-independent mechanisms. The protein is composed of three domains (N-terminal domain, midregion, and Cterminal domain).5 The C-terminal domain contains an 18-basic amino acid motif (Figure


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1) defined by amino acid residues 215-232 of mature IGFBP-3 previously shown to bind heparin and certain glycosaminoglycans, including hyaluronan (HA).5,8,20–22 Upon closer inspection of this region, two overlapping sequences can be identified (Figure 1, underlined) that contain the [B(X7)B] motif previously reported to be necessary to bind HA, where “B” can be either arginine or lysine, and “X” any non-acidic amino acid.23

A

215

KKGFYKKKQCRPSKGRKR

232

B

Humanin

1MAPRGFSCLLLLTSEIDLPVKRRA24

Figure 1. The peptide humanin binds to the same sequence on IGFBP-3 containing the HA binding motif. A. Putative HA-binding motif of IGFBP-3. B. Humanin peptide sequence.

Under normal physiological conditions, there is tight regulation between cells and extracellular matrix (ECM) components. That regulation is dramatically disturbed during tissue injury and remodeling in human diseases, resulting in loss of cellular communication.24,25 HA is an anionic glycosaminoglycan polymer composed of a simple


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Biochemistry

disaccharide sequence (D-glucuronic acid and D-N-acetylglucosamine) without any known post-synthetic modification.26–29 It is synthesized by enzymes localized at the cell membrane and it is largely abundant extracellularly. It is a major component of the ECM and via interactions with its binding proteins, is associated with the rapid matrix remodeling that occurs during the pathogenesis of several human diseases. HA binding to its main receptor, CD44, is thought to range in affinity30–34 and is known to promote cell survival pathways.35 HA is a dynamic molecule of various sizes ranging from high molecular weights (HMW-HA, 1–10 million Da) to oligosaccharides that can all modulate receptor activation.26,27

Humanin, a small mitochondrial-derived peptide discovered by the Nishimoto Lab9,36,37, is known to bind with high affinity and specificity to the 18-amino acid residue (215-232) heparin-binding domain of IGFBP-338 that also binds HA.

It is encoded in the

mitochondrial genome by an open reading frame in the 16S rRNA region 39–43 and composed of 24 amino acids (Figure 1) when translated in the cytoplasm and 21 amino acids when translated in the mitochondria.41 Both of these humanin forms have been shown to exhibit antiapoptotic effects.44 Growing evidence suggests that humanin is a strong and potent cyto- and neuro-protective peptide against a range of stresses and in disease models36,40–42,45–50 including Huntington's, stroke, and Alzheimer's disease models, memory loss, inflammation, and type 2 diabetes. In response to cellular stress, humanin needs to be secreted to exhibit diverse extracellular signaling functions and broad cytoprotective effects in various diseases.36,38 It is known to be regulated by growth hormone and IGF-I and in turn, regulates IGF-I likely


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through binding to the C-terminal region of IGFBP-3.9 Humanin levels have been measured in cerebrospinal fluid, plasma, and seminal fluid 9,42 and its expression has been detected in many tissues that include liver, fat, skeletal muscle, and hypothalamus.51 However, it can act via both intracellular binding partners and extracellular receptor mechanisms with different modes of action.9,40–42 Extracellularly, humanin was found to directly interact with and activate FPRL1/2,41,52,53 G protein-coupled formylpeptide-like-1 receptors.

It was also reported to exhibit its protective properties via extracellular

interactions with a tripartite cytokine-like receptor complex composed of the CNTF receptor, WSX1 receptor, and gp130 and the downstream signaling pathway, JAK2– STAT-3.42 Intracellularly, it exhibits anti-apoptotic effects via interaction with pro-apoptotic proteins such as Bax and tBID.41 We have recently synthesized peptides to show that humanin binds to IGFBP3 and interferes with the binding of importin-β1 to IGFBP3 in vitro, suggesting a possible mechanism of action for the peptide in regulating cell survival as an inhibitor of IGFBP3 nuclear translocation in certain cell types.54 Humanin has been shown to antagonize or be synergistic with IGFBP-3 functions. For example, in testicular germ cells, administration of humanin blocked hormone deprivation induced apoptosis, while opposite effects were observed with IGFBP-3 treatment.55 Humanin was also shown to interact with the C-terminal domain of IGFBP-3, blocking IGFBP-3 induced cell death, without affecting or competing with binding of IGF-I to IGFBP-3.9 Conversely, synergistic protective effects of IGFBP-3 with humanin were shown in protection against Aβ1-43 toxicity in mouse primary neurons.38


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Biochemistry

Characterization of Protein-peptide-carbohydrate interactions is of

paramount

importance for understanding human disease since these interactions mediate numerous cellular and biochemical processes.

This study examined overlapping interactions

between the IGFBP-3 protein, the humanin peptide, the carbohydrate, HA, and its receptor, CD44. We show that both HA and humanin compete for binding to the 18-basic amino acid residue sequence of IGFBP-3, thus, regulating HA-CD44 binding. Better understanding of these interactions may provide grounds for novel approaches in deciphering mechanisms employed by IGFBP-3 and humanin in regulating HA-CD44 signaling, advance our knowledge of diseases resulting from dysregulation of proteinpeptide-carbohydrate interactions, and provide insights into novel therapeutic strategies to treat those diseases.

MATERIALS AND METHODS Materials. Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids and Obenzotriazolyl-N,N,N',N'-tetramethyluronium

hexafluorophosphate

(HBTU)

were

purchased from Anaspec Inc. Dichloromethane was purchased from Acros Organics. Dimethylformamide (DMF) and HPLC-grade acetonitrile (ACN) were from EMD Chemicals, Inc. Piperidine, triisopropylsilane (TIS), diethyl ether, ethanol, phenol, acetic anhydride, trifluoroacetic acid (TFA), Phosphate Buffer Saline (PBS), and nitrocellulose membranes were purchased from Sigma-Aldrich. Rink amide MBHA resin was purchased from Nova Biochem.

Humanin (018-26, B-018-26, UniProt

accession ID: Q8IVG9) was purchased from Phoenix Pharmaceuticals. The humanin mutant (F6A-humanin, MAPRGASCLLLLTSEIDLPVKRRA) was synthesized by the


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RS synthesis company. Recombinant human IGFBP3 protein (YCP1009, UniProt accession ID: P17936) was from Speed BioSystems. HA (GLR002) and recombinant human CD44-Fc chimera protein (3660, UniProt accession ID: E9PKC6), were purchased from R&D Systems.

HA-biotin (B1557) and streptavidin-horseradish

peroxidase (HRP) conjugate were purchased from Sigma-Aldrich. Rabbit polyclonal humanin antibody (56876) and goat anti-rabbit IgG secondary antibody (HRP) were purchased from Novus Biologicals.

IGFBP3 mouse monoclonal antibody (sc-

374365), goat anti-mouse IgG-HRP (sc-2005), and protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology.

Sheep polyclonal anti-HA antibody

(ab53842) and rabbit anti-sheep IgG (ab6747) were from Abcam. CD44 antibody (5F12) (MA5-12394), mouse IgG isotype control, (mIgG), mouse monoclonal antihuman IgG Fc secondary antibody (HRP, 05-4220), 3,3’,5,5’-tetramethylbenzidine, and the IGFBP-3 ELISA kit (EHIGFBP3) were from ThermoFisher. BCA protein assay kit and the super signal west Pico luminol (chemiluminescence) reagent were from Pierce. The humanin ELISA kit (KTE61475) was purchased from Abbkine.

Solid

Phase

Peptide

Synthesis.

215KKGFYKKKQCRPSKGRKR 232

A

peptide

corresponding

to

residues

of IGFBP-3 was synthesized by solid phase peptide

synthesis on a PS3 synthesizer from Protein Technologies, using rink amide MBHA resin as a solid support on a 0.1 mmole scale. The side chains of Tyr and Ser were protected as the t-butyl derivatives, Lys as t-butyloxycarbonyl (Boc), Gln and Cys as the trityl (Trt) forms, and Arg as the 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (Pmc) form. N-alpha-Fmoc protected amino acids were coupled in four-fold excess


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Biochemistry

using HBTU as an activating agent, and 20% piperidine was used for deprotection. The peptide was cleaved from the resin by stirring in a 10 mL cocktail consisting of 5% distilled water, 5% phenol scavenger, 2% TIS, and 88% TFA for 2 hours at room temperature. The peptide was precipitated with cold diethyl ether, filtered, dissolved in 35% ACN/H2O and lyophilized. The crude peptide was purified by reversed-phase HPLC on a Phenomenex C18 column (25cm x 2.2cm), using 0.1% TFA in water (solvent A) and 0.1% TFA in ACN (solvent B), with a gradient of 10 to 50% B over 2 hours at a flow rate of 10 mL/min. Purity was assessed by analytical HPLC on a Phenomenex C18 column (25cm x 4.6mm), at 220 nm. The molecular weight was determined to be 2221.330 g/mol using paper spray ionization mass spectrometry. The peptide was dissolved in 10% DMSO and PBS buffer, pH 7.4, to a final concentration of 1 mg/mL.

Cell Culture. Human lung adenocarcinoma epithelial cells (A549 cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Approximately 1 x 10 5 cells were seeded in 25 cm2 tissue culture flasks in 5 mL HyClone Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) (GE Healthcare Life Sciences, Pittsburgh, PA), supplemented with 10% Fetalgro bovine growth serum (FBS, RMBIO, Missoula, MT), 50 U/mL penicillin, and 50 U/mL streptomycin (Invitrogen Life Technologies, Carlsbad, CA) and allowed to grow overnight in an incubator at 37 oC, 95% humidity, and 5% CO2. The cells were counted using a hemocytometer after trypan blue staining.


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Co-Immunoprecipitation. The binding of humanin to IGFBP-3 in media of A549 cells was investigated by co-immunoprecipitation. Cells were grown as described above then washed with PBS and cultured in serum-free media for 48 hours. The media was collected and protein concentrations in the media were determined using the BCA protein assay kit. Equivalent amounts of total protein were used for all samples analyzed. Humanin was immunoprecipitated from 300 µL conditioned media (0.5 µg protein/µL) of A549 cells, 2 days post serum starvation using humanin specific antibodies, then IGFBP-3 in the beads and that left in the supernatant was detected by western blotting using anti-IGFBP-3 antibodies as described below. Conversely, IGFBP-3 was immunoprecipitated with anti-IGFBP-3 antibodies and humanin in the beads and supernatant was visualized with humanin specific antibodies. For each sample analyzed, 250 µL of Protein A/G PLUS-agarose beads were prepared (150 µL for preclearing the sample, and 100 µL for immunoprecipitation). The beads were washed three times in Tris-Buffered Saline and Tween 20 (TBST). The media (350 µL) was precleared by adding 150 µL of the beads and the mixture placed on a shaker for 50 minutes at room temperature. Following centrifugation at 3000 xg for 1 minute, the beads were discarded, and the precleared media was transferred to an Eppendorf tube. Beads (100 µL in TBST) were then added to the precleared media (300 µL) along with 10 µg of primary antibody and allowed to incubate on a shaker at 4oC overnight. Following centrifugation at 3000 xg for 1 minute, the supernatant and bead fractions were collected and analyzed by western blotting. Media not incubated with cells (300 µL) and TBST (300 µL) were used as a blank.


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Biochemistry

Western Blot Analysis. Following immunoprecipitation, samples were boiled in 1X SDS, loaded and separated by SDS-PAGE on a 20% gel then transferred to a nitrocellulose membrane. The membrane was blocked in TBST buffer, pH 7.6 containing 5% nonfat milk for 6 hours at 4 oC. The membrane was then incubated with the specific primary antibody in the blocking buffer, diluted as specified by the manufacturer at room temperature overnight with gentle shaking. After washing three times with TBST, the membrane was incubated with a HRP labeled secondary antibody

in

the

recommendation.

blocking

buffer,

diluted

according

to

the

manufacturer’s

Subsequent to washing three times in TBST, the blots were

developed using super signal west pico luminol (chemiluminescence) reagent and imaged with a Bio-Rad molecular imager.

ELISA. Humanin and IGFBP-3 peptide were dissolved in 10% DMSO and PBS buffer, pH 7.4, to a final concentration of 1 mg/mL. CD44 and HA were reconstituted to 100 μg/mL in sterile PBS and IGFBP-3 was reconstituted with water to 400 µg/mL. Wells of a Nunc MaxiSorpTM 96-well Flat Bottom plate (ThermoFisher) were coated with 100 µL of 25–200 nM protein/peptide in sample buffer (15 mM Na 2CO3, 50 mM NaHCO3, pH 9.6), and incubated overnight at 4°C on a shaker to allow the protein/peptide to bind to the plate. Following incubation, the wells were washed 4x with TBST, filled with 400 µL blocking buffer (110 mM KCl, 5 mM NaHCO3, 5 mM MgCl2, 1 mM EGTA, 0.1 mM CaCl2, 20 mM HEPES, 1% BSA, pH 7.4), and incubated overnight at 4°C on a shaker. After washing 4x with TBST, 100 µL PBS buffer containing the desired concentration of sample were added to each well, and the plate incubated overnight at 4°C on a shaker to allow


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interaction of the sample with the protein/peptide bound to the plate. The wells were then washed 4x with TBST before proceeding in one of two ways: 1) If analyzing biotinylated samples, 100 µL streptavidin-HRP conjugate in TBST (1:2500 dilution) were added before incubating for 3 hours at room temperature on a shaker, or 2) when analyzing samples without biotin, 100 µL TBST containing the necessary primary antibody were added at the manufacturer’s recommendation, incubated for 3 hours at room temperature on a shaker, and washed again 4x with TBST. TBST (100 µL) containing the secondary antibody were then added at the manufacturer’s recommendation and incubated for 1 hour at room temperature on a shaker. For either sample type, the plate was then washed 5x with TBST followed by the addition of 100 µL 3,3’,5,5’-tetramethylbenzidine resulting in a blue color change. After incubating at room temperature for 0.5 – 15 minutes, the reaction was stopped with 100 µL 2M H2SO4, resulting in a yellow color change, and the absorbance measured at 450 nm. Statistical analysis was determined by the GraphPad Prism 7.04 software using nonlinear regression and a dose response curve fit. Three to five independent experiments were carried out for each assay condition.

Dot Blotting. Binding of IGFBP-3 to HA was measured as described previously56,57 but with few modifications. It is known that HA alone does not bind to nitrocellulose but will bind only in the presence of proteins or peptides that bind to it.58 Purified IGFBP-3 was mixed with different concentrations of HA for 2 hours at room temperature, then spotted onto a nitrocellulose membrane. The membrane was allowed to dry then non-specific sites were blocked by soaking the blot in TBST containing 5% BSA in a 10 cm Petri dish for one hour at room temperature. The blot was next incubated with HA-sheep polyclonal


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Biochemistry

antibodies in BSA/TBST according to the manufacturer’s instructions overnight at room temperature. The membrane was then washed three times with TBST (3 x 5 minutes) then incubated with rabbit polyclonal to Sheep IgG - H&L (HRP) secondary antibody following the manufacturer's recommendation for 30 minutes at room temperature. The membrane was next washed three times with TBST (3 x 5 minutes), then once with TBS for 5 minutes. The HA bound to IGFBP-3 and retained on the filter membrane was detected using super signal west pico luminol (chemiluminescence) reagent and imaged with a Bio-Rad molecular imager. Distilled water incubated with HA was used as a negative control.

MTT Assay. The MTT reduction assay, used to measure cell viability, was purchased from Sigma-Aldrich. A549 cells were seeded in 96-well plates at 0.2 × 105 cells per well in 200 μL 10% FBS-supplemented media as described above and maintained overnight at 95% humidity and 5% CO2. The media from the microplate was then removed and 200 μL serum-free media was added. The cells were allowed to incubate for 12 hours. The cells were then treated with control mIgG (5 μg/mL) or with 5 μg/mL CD44 antibody (5F12) for 2 hours prior to addition of and incubation with 50 nM IGFBP-3 and/or 50 or 150 nM humanin for 48 hours.

Media containing the specific components in the different

treatments was replaced every 12h. One set of wells was reserved for a negative control, which contained only media with DMSO. The final concentration of DMSO in each well never exceeded 0.1%. Following a 48 hours incubation, the cells were incubated with MTT (0.5 mg/mL) for 4 hours after which the media was carefully removed. MTT assays were carried out in the dark due to sensitivity of the MTT reagent. The formazan crystals


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were dissolved in 100 μL DMSO and the absorbance was measured in a plate reader at 570 nm. Positive controls included untreated cells while DMSO alone represented a negative control. Statistical analysis was conducted with GraphPad Prism version 7.04 for Windows. Significant values were considered at p < 0.05 and highly significant values at p < 0.01 compared with the control.

RESULTS AND DISCUSSION

The IGFBP-3 Protein and the 18-Residue IGFBP-3 Peptide, Bind HA.

Because

IGFBP-3 contains a HA-binding motif (Figure 1) and since the protein was previously reported to bind certain glycosaminoglycans including HA,20–22 a dot blot was performed taking advantage of the fact that HA will only bind to nitrocellulose in the presence of proteins or peptides that bind to it.57,58 IGFBP-3 was mixed with HA then applied onto a nitrocellulose membrane. The free HA was then washed away, and the HA bound to IGFBP-3 and retained on the nitrocellulose membrane was visualized (Figure 2A) as described in the Materials and Methods.

As expected, no HA was bound to the

membrane when distilled water was used, however, upon incubation with IGFBP-3, HA was detected in a complex (Figure 2A, lanes 2-3). In order to examine whether the 18amino acid residue basic sequence in the C-terminal domain of IGFBP-3 that contains the HA binding motif (Figure 1) can independently bind HA and to compare the affinity of that binding to that of the full-length protein, a peptide corresponding to amino acid residues 215-232 was synthesized. ELISAs were carried out and showed that IGFBP-3 protein and peptide bound HA with a Kd of 189 ± 4 nM or 258 ± 6 nM, respectively (Figure


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Biochemistry

2B). These results clearly show that the 18-amino acid residue peptide is able to bind HA with a comparable affinity to that found for the full-length protein and that the HA binding site is largely contained within this basic region of IGFBP-3. We then carried out a competitive ELISA and found that (Figure 2C) the full-length IGFBP-3 protein is able to compete with the IGFBP-3 peptide for binding to biotinylated-HA with an IC50 of 48 ± 1 nM. These results clearly suggest that HA can bind to amino acid residues 215-232 of IGFBP-3 and that the full-length IGFBP-3 protein is effective at competing with its peptide for binding to HA.

In support of this suggestion, this C-terminal domain was previously reported to exhibit an approximate 4-fold higher affinity for heparin compared to an internal heparin binding domain.20 Moreover, synthetic peptides corresponding to this 18-amino acid residue sequence were shown22 to bind directly to the endothelial cell extracellular matrix and to inhibit binding of IGFBP-3 to endothelial cell monolayers. This region alone was reported to be likely sufficient to bind HA and other components of the extracellular matrix that can act to block access of the full-length IGFBP-3 protein to endothelial cell monolayers.


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Biochemistry

A

1 P e rc e n t O p tic a l D e n s ity

B

2

3

100

50

IG F B P -3 IG F B P -3 P e p tid e 0 0

500

1000

B io tin - H A (n M )

C 100

P e rc e n t O p tic a l D e n s ity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 42

50

0 1

10

100

1000

IG F B P 3 (n M )

Figure 2. IGFBP-3 and peptide bind HA. A. A dot-blot was performed where IGFBP-3 (1.62 µM) was incubated with 0.5 µM HA (lane 2), and 1.75 µM HA (lane 3) for 2 hours at room temperature then spotted in triplicates onto a nitrocellulose membrane. Lane 1 represents a blank with distilled water and 1.75 µM HA. The free HA was washed away, and the HA bound to IGFBP-3 and retained on the filter membrane, was detected using HA-sheep polyclonal antibodies and rabbit polyclonal to Sheep IgG - H&L (HRP) secondary antibody as described in the Materials and Methods. B. IGFBP-3 (50 nM) or peptide (50 nM) were bound to the wells then increasing concentrations of biotinylated-HA were added and processed as described in the text (n= 3). Optical density measurements were normalized by expressing each point in relation to the best-fitted Emax value (set to 100%) and then plotting them with GraphPad Prism 7.04. The equilibrium dissociation constants were determined by fitting the data to a single binding site model. IGFBP-3 protein and peptide bound HA with a Kd of 189 ± 4 nM or 258 ± 6 nM, respectively. C. IGFBP-3 peptide (50 nM) was bound to the wells then biotinylated-HA (400 nM) was added along with increasing concentrations of the full-length IGFBP-3 protein and processed as described in the text. Data are expressed as the mean ± S.D. of three independent experiments. The optical density measurements were normalized and then plotted as a function of increasing IGFBP-3 concentrations with a nonlinear regression curve fitting approach using GraphPad Prism 7.04. The dashed line indicates 50% of maximum binding. The IC50 was determined to be 48 ± 1 ACS Paragon Plus Environment nM.

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Biochemistry

IGFBP-3 Is More Efficient at Blocking the Binding Of IGFBP-3 Peptide to Humanin Than HA. Elegant early studies by Ikonen et al.38 have shown that the 18-amino acid heparin-binding domain of IGFBP-3 (215KKGFYKKKQCRPSKGRKR232) is the binding site for humanin. This 18-residue peptide was found to block humanin binding to 125I-IGFBP3 to a similar extent as full-length unlabeled IGFBP-3 suggesting that the 18-basic residue sequence in the C-terminal region of IGFBP-3 is the primary binding site for humanin. Consistent with these reports, we found that IGFBP-3 binds to humanin with a Kd of 101 ± 3 nM (Figure 3A) while the peptide bound with a comparable affinity of 120 ± 3 nM. These results are consistent with this 18-amino acid residue region being the primary site on IGFBP-3 that binds humanin. We next examined the ability of the IGFBP-3 protein to compete with the peptide for binding to humanin by first binding the IGFBP-3 peptide (50 nM) to the ELISA plate wells followed by incubation with 300 nM biotinylated humanin in the absence and presence of increasing concentrations of the IGFBP-3 protein (Figure 3B).

The concentration of the protein indicated by the arrow on the x-axis that

corresponds to 50% inhibition was found to be 61 ± 2 nM. Since HA was found to bind both the IGFBP-3 protein and peptide with comparable affinity (Figure 2), we wanted to examine whether HA is able to compete with humanin and block its access to the IGFBP3 peptide (Figure 3C). The IGFBP-3 peptide (50 nM) was bound to the wells then 300 nM biotinylated humanin was added along with increasing concentrations of HA (Figure 3C). While HA was able to compete with and block humanin from binding to the IGFBP3 peptide, it was less efficient in doing so than the IGFBP-3 protein (Figure 3B) with an IC50 of 183 ± 5 nM. These results suggest that HA and humanin compete for binding to the 18-residue sequence on IGFBP-3 but that HA is less efficient at blocking humanin


17

Biochemistry

binding to the IGFBP-3 peptide, a result that might reflect the lower binding affinity of HA to the IGFBP-3 peptide (Figure 2B, 258 ± 6 nM) as compared to that of the IGFBP-3 protein to humanin (Figure 3A, 101 ± 3 nM).


A 100

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Page 18 of 42

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Figure 3. IGFBP-3 competes with the binding of the IGFBP-3 peptide to humanin more efficiently than HA. A. IGFBP-3 (50 nM) or peptide (50 nM) was bound to the wells, then increasing concentrations of biotinylatedhumanin were added and processed as described in the text (n= 3). Optical density measurements were normalized by expressing each point in relation to the best-fitted Emax value (set to 100%). The curve was drawn using GraphPad Prism 7.04. IGFBP-3 protein and peptide bound to humanin with a Kd of 101 ± 3 nM or 120 ± 3 nM, respectively. The equilibrium dissociation constants were determined by fitting the data to a single binding site model. For B and C, IGFBP-3 peptide (50 nM) was bound to the wells. Biotinylated-humanin (300 nM) was then added along with increasing concentrations of full-length IGFBP-3 (B) or HA (C) and processed as described in the text. Data are expressed as the mean ± S.D. of three independent experiments. The IGFBP-3 protein concentration indicated by the arrow on the x-axis that corresponds to 50% inhibition was found to be 61 ± 2 nM. The IC50 for HA was found to be 183 ± 5 nM. For B and C, optical density measurements were normalized and then plotted as a function of increasing IGFBP-3 or HA concentrations with a nonlinear regression curve fitting approach using GraphPad Prism 7.04. The dashed lines indicate 50% of maximum binding.


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Biochemistry

Exogenous Humanin and IGFBP-3 Can Co-immunoprecipitate From Human Alveolar Basal Epithelial (A549) Cell Media. It is known that in A549 cells, IGFBP-3 is present in the extracellular compartment and is barely detectable in the intracellular compartment.59 In addition, humanin is mainly secreted and has been shown to circulate in the plasma in association with IGFBP-3.60,61 To test whether exogenous IGFBP-3 and humanin interact in vivo, A549 cells (1 X 105) were seeded in regular Dulbecco’s Modified Eagle medium (Materials and Methods) and allowed to grow overnight in an incubator at 37 oC and 5% CO 2. Following washing the cells with PBS, they were cultured in serum-free media for 48 hours. The media (0.5 µg protein/µL) was then collected and examined for the binding of IGFBP-3 and humanin. Using humaninspecific antibodies, humanin was immunoprecipitated from the media then IGFBP-3 visualized using IGFBP-3-specific antibodies. Compared to the negative control (Figure 4A, lane 1) where media not incubated with cells was used as a blank, IGFBP-3 was found to immunoprecipitate with humanin in the beads (Figure 4A, lane 2). Similarly, humanin was found in a complex with IGFBP-3 (Figure 5A, lane 2) upon IGFBP-3 immunoprecipitation using anti-IGFBP-3-specific antibodies. It is unclear why there are two bands recognized by the anti-humanin antibodies, but it may reflect some form of a posttranslational modification since the lowest band of about 3 kDa is expected for humanin. Moreover, while the 20% SDS-gels used for the western blots (Figures 4, 5) normally showed IGFBP-3 as a more single species, diffuse bands of the protein were observed when lower percentage SDS-gels were used. The extent of band diffusion also depended on how long the protein gels were run. The shorter the time the gels were run, the more single the band appeared. These diffuse bands are likely due to the observation


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Page 20 of 42

that secreted IGFBP-3 is heavily glycosylated.22,62,63 To further verify these interactions, anti-humanin specific antibodies were bound (1:100 dilution) to ELISA wells (Figure 4B). Following the blocking step, 300 µL media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation, was added and the IGFBP-3 was detected using IGFBP-3 specific antibodies (Figure 4B, lane 2). Compared to media not incubated with cells (Figure 4B, lane 1), there was an approximate 4.5-fold increase in the amount of IGFBP-3 bound to humanin (Figure 4B, lane 2) from the 48 hours post serum starvation media. ELISA also showed an approximate 4.2-fold increase in the amount of humanin found in a complex with IGFBP-3 when wells were coated with anti-IGFBP-3 antibodies (Figure 5B). These results show that humanin and IGFBP-3 can be found in a complex in media collected from A549 cells 48 hours post serum starvation.


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Biochemistry

3

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Figure 4. IGFBP-3 is co-immunoprecipitated in a complex with humanin from media of A549 cells. A. Humanin was immunoprecipitated from 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation, with anti-humanin specific antibodies. IGFBP-3, co-immunoprecipitated with humanin, was then visualized by western blotting using IGFBP-3-specific antibodies. Lane 1 is a blank using media not incubated with cells, lanes 2 and 3 show IGFBP-3 found in the beads and left in the supernatant, respectively. B. Anti-humanin specific antibodies were added (1:100 dilution) to ELISA wells. The wells were blocked, then 300 µL of media not incubated with cells (lane 1) and 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hour post serum starvation (lane 2) were added and the IGFBP-3 was detected using anti-IGFBP-3 specific antibodies then processed as described in the Materials and Methods. Each column represents the mean ± S.D. of three independent experiments.


21

Biochemistry

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Page 22 of 42

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Figure 5. Humanin co-immunoprecipitates with IGFBP-3 from media of A549 cells. A. Using anti-IGFBP-3 specific antibodies, the protein was immunoprecipitated from 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation. Humanin co-immunoprecipitated with IGFBP-3 was then visualized by western blotting using anti-humanin specific antibodies. Lane 1 is a blank using media not incubated with cells, lanes 2 and 3 show humanin found in the beads and left in the supernatant, respectively. B. IGFBP-3 specific antibodies were added (1:100 dilution) to ELISA wells. Following the blocking step, 300 µL of media not incubated with cells (lane 1) and 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation (lane 2) were added and the humanin was detected using anti-humanin specific antibodies then processed as described in the Materials and Methods section. Each column represents the mean ± S.D. of three independent experiments.


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Biochemistry

Only IGFBP-3 Not Bound to Humanin Can Be Found in A Complex With HA. The results in Figure 2 show that purified IGFBP-3 binds HA. To test whether exogenous IGFBP-3 from A549 cell media harvested 48 hours post serum starvation could bind HA, an ELISA was carried out (Figure 6A) where HA (400 nM) was bound to ELISA wells overnight. The wells were blocked then incubated with 300 µL media that has not been incubated with cells (Figure 6A, lane 1) and with 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation (lanes 2-6). Using anti-IGFBP-3 mouse monoclonal primary antibodies and goat anti-mouse IgG-HRP secondary antibodies, IGFBP-3 was detected to bind immobilized HA with an approximate 4.9-fold increase over the control (Figure 6A, lane 2). We then examined whether IGFBP-3 bound to HA immobilized in the wells is also complexed with humanin. Using anti-humanin rabbit polyclonal primary antibodies and goat anti-rabbit IgG secondary antibodies (HRP), no humanin was detected (Figure 6A, lane 3). When both IGFBP-3 and humanin primary and secondary antibodies were used (Figure 6A, lane 4), no further increase in absorbance was observed compared to using only IGFBP-3 antibodies (Figure 6A, lane 2). There was a 4.8-fold increase over the control, identical to that found upon using only anti-IGFBP-3 antibodies. No signal compared to the control (lane 1) was observed when both primary antibodies were used without the corresponding secondary antibodies (lane 5), or with both the secondary but not the primary antibodies (lane 6). The sequence of IGFBP-3 (215KKGFYKKKQCRPSKGRKR232) appears to be the primary region of the protein at the C-terminal end (Figures 2,3) that binds either HA or humanin, although the residues in IGFBP-3 responsible for binding HA and humanin may not be identical, only that they both fall, at least in part, within the 18-residue basic domain. It is


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Page 24 of 42

possible that anti-humanin antibodies are unable to access humanin bound to IGFBP-3 when the IGFBP-3 is associated with immobilized HA. Alternatively, a likely interpretation of the results in Figure 6A is that while IGFBP-3 and humanin can be found in a complex (Figures 4,5), IGFBP-3 binds immobilized HA through its 215-232 region, rendering it inaccessible to humanin. Further support for this interpretation was obtained when a lack of dose-dependent signal was observed when 400 nM HA was immobilized to the wells, incubated with 50 nM IGFBP-3 prior to addition of increasing concentrations of biotinylated humanin (Figure 6B). Binding of increasing concentrations of biotinylated humanin with IGFBP-3 likely dislodged the protein from binding to immobilized HA resulting in lack of a measurable signal.


24

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Biochemistry

0 .8

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Figure 6. A. The IGFBP-3 fraction from A549 cell media that binds HA, is not complexed with humanin. HA (400 nM) was allowed to bind to ELISA wells overnight. The wells were blocked then incubated with 300 µL media not incubated with cells (lane 1), and with 300 µL conditioned media (0.5 µg/µL) of A549 cells, 48 hours post serum starvation (lane 2-6). Antibodies were then added to the wells as follows: anti-IGFBP-3 antibodies to detect IGFBP-3 (lane 2), anti-humanin antibodies to detect humanin (lane 3), both IGFBP-3 and humanin primary antibodies along with their corresponding secondary antibodies (lane 4), both primary antibodies without the secondary antibodies (lane 5), and both the secondary antibodies only (lane 6). Each column represents the mean ± S.D. of three independent experiments. B. HA (400 nM) was bound to the wells then incubated with 50 nM IGFBP-3 prior to adding increasing concentrations of biotinylated humanin and processed as described in the text. Data were subtracted from a blank containing all ingredients but without IGFBP-3.


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Page 26 of 42

CD44 Competes with the Binding of HA to IGFBP-3. The interaction of HA with CD44 is well-established.24,25,27,28,64 To determine the binding affinity under our conditions, HA (50 nM) was bound to the wells, then increasing concentrations of CD44-Fc were added and the signal processed as described in the Material and Methods. The Kd was found to be 62 ± 4 nM (Figure 7A). This value is similar to those reported earlier.65 No binding was observed when 200 nM of either humanin or IGFBP-3 in the absence of HA were bound to the wells and incubated with increasing concentrations of CD44-Fc. We next examined whether CD44 is able to bind HA and block its interactions with IGFBP-3. IGFBP-3 (50 nM) was bound to the wells then biotinylated-HA was added at concentrations below the Kd (100 nM) and above the Kd (400 nM) along with increasing concentrations of CD44. The CD44 concentration that corresponds to 50% inhibition was found to be 42 ± 2 nM when 100 nM of HA was used, as compared to 223 ± 11 nM when 400 nM HA was added (Figure 7B). These results indicate that at more saturating concentrations of HA, higher concentrations of CD44 are required to block binding of HA to IGFBP-3. We then examined (Figure 7C) whether CD44 can interfere with the binding of IGFBP-3 to humanin. IGFBP-3 protein (50 nM), or IGFBP-3 peptide (50 nM) were bound to the wells then biotinylated-humanin (400 nM) was added along with increasing concentrations of CD44. No effect on humanin interaction with either IGFBP-3 protein or peptide was observed upon addition of increasing concentrations of CD44 demonstrating that binding of CD44 is specific to HA. Similar results were obtained when we tested the effects of using lower concentrations of biotinylated humanin (50 nM) (Figure 7D) which would give the assay greater sensitivity to the possibility that CD44 can displace humanin from IGFBP-3.


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Biochemistry

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Figure 7. Binding of HA to IGFBP-3 is inhibited by CD44. A. HA (50 nM) was bound to the wells then increasing concentrations of CD44-Fc (●) were added and the signal processed as described in the text (n= 3). The Kd value, determined by fitting the data to a one site binding model in Graphpad prism 7.04 with a nonlinear regression curve fitting approach, was found to be 62 ± 4 nM. As a control, humanin (▄, 200 nM), or IGFBP-3 (♦, 200 nM), were bound to the wells and incubated with CD44-Fc. The data were normalized to controls incubated with BSA and fold change relative to the control was calculated and fit with a nonlinear regression curve using GraphPad Prism 7.04. B. IGFBP-3 (50 nM) was bound to the wells. Biotinylated-HA, 100 nM (●) and 400 nM (▄) were then added along with increasing concentrations of CD44 and processed as described in the text (n= 4). Arrows on the x-axis indicate the CD44 concentration that corresponds to 50% inhibition for each curve (●, 42 ± 2 nM), (▄, 223 ± 11 nM). The dashed line indicates 50% of maximum binding. C, D. IGFBP-3 protein, (●, 50 nM), or IGFBP-3 peptide (▄, 50 nM) were bound to the wells. Biotinylated-humanin (400 nM, C) or (50 nM, D) was then added along with increasing concentrations of CD44 and processed as described in the text. Data are expressed as the mean ± S.D. of three independent experiments. For B and C, optical density measurements were normalized by plotting mean absorbances for each concentration as a fraction of the maximal binding (set to 100%), then plotting them as a function of CD44 concentrations.


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Page 28 of 42

Humanin Binds To IGFBP-3 Allowing Unimpeded Access of CD44-Fc to Immobilized HA. Our results show that HA binds to the IGFBP-3 protein and peptide (Figure 2) and to CD44 (Figure 7) and that humanin binds to IGFBP-3 and IGFBP-3 peptide (Figure 3). We next added IGFBP-3 or IGFBP-3 peptide along with CD44-Fc to HA bound to ELISA wells.

Detection of bound CD44 increased with increasing

concentrations of WT-humanin (Figure 8) presumably since the humanin peptide binds to the IGFBP-3 protein or the IGFBP-3 peptide freeing HA to bind to CD44-Fc. The absorbance was found to be at maximum with concentrations of humanin that exceed 400 nM, a result consistent with those found in Figure 3 and suggests that most if not all of the IGFBP-3 or peptide is complexed with humanin and not with HA at these concentrations allowing unimpeded access of CD44-Fc to immobilized HA.

No signal

was obtained when a humanin mutant, F6A, demonstrated earlier38 to completely abolish the interaction between humanin and IGFBP-3, was used (Figure 8). This result suggests that IGFBP-3 remained bound to HA as it lacks the ability to bind mutant humanin, thus blocking CD44-Fc from binding to HA.


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Biochemistry


Page 29 of 42

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Figure 8. Increasing concentrations of WT-humanin, but not the F6A-humanin mutant, sequesters IGFBP-3 away from HA allowing CD44-Fc to bind immobilized HA. HA (25 nM) was bound to the wells followed by the addition of IGFBP-3 protein (●, 50 nM) or IGFBP-3 peptide (▄, 50 nM), CD44-Fc (10 nM) and with increasing concentrations of WT-humanin. The same experiment but with increasing concentrations of the F6A-humanin mutant (50 nM IGFBP-3 protein, ▼) and (50 nM IGFBP-3 peptide, ▲) is also shown. Data were processed as described in the text and expressed as the mean ± S.D. of three independent experiments. Optical density measurements were normalized by expressing each point in relation to the best-fitted Emax value (set to 100%) and then plotting them as a function of humanin concentrations using a nonlinear regression curve fitting approach with GraphPad Prism 7.04.


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Page 30 of 42

The Cytotoxic Effects of IGFBP-3 on A549 Cell Viability Depend on HA-CD44 Interactions and are Blocked by Humanin. To test whether our in vitro data can be demonstrated in cells, 0.2 × 105 A549 cells were grown in 10% FBS-supplemented media overnight then the cell monolayers were incubated in serum-free medium for 0, 12, and 48 hours. Aliquots of the conditioned media were collected at 0, 12, and 48 hours post serum starvation then tested for the presence of IGFBP-3 using a human IGFBP-3 ELISA kit and the presence of humanin using a humanin ELISA kit. The amount of IGFBP-3 and humanin were quantitated using a standard curve of known concentrations following the manufacturers’ recommendation. Measurements were normalized over the total protein concentration in the media. While no IGFBP-3 or humanin was detected at 0 or 12 hours post serum starvation, the concentration of IGFBP-3 and humanin in the media 48 hours post serum starvation was determined to be 1520 ng/mL and 145 ng/mL, respectively. To avoid complications of interpreting the data by the already secreted IGFBP-3 and humanin in A549 media 48 hours post serum starvation, cells were seeded in 96-well plates at 0.2 × 105 cells per well in 10% FBS-supplemented media then serum starved for 12 hours. The cells were then treated with mouse IgG isotype control with no relevant specificity to a target antigen, (mIgG, 5 μg/mL), or 5 μg/mL anti-CD44 antibody (5F12) known to be antagonistic towards HA-CD44 molecular interactions, for 2 hours prior to addition of either 50 nM IGFBP-3, humanin (50 nM, 150nM), or in combination (Figure 9). The concentrations of added IGFBP-3 or humanin of 50 nM were chosen to be close to the values measured by the ELISA in the conditioned media. The cells were then allowed to incubate for 48 hours prior to assessing cell viability by the MTT assay. Media containing the specific components in the different treatments was replaced every 12


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Biochemistry

hours to ensure the absence of secreted IGFBP-3 or humanin during the assay. As predicted, cell viability was decreased in the presence of the anti-CD44 5F12 antibodies that block HA-CD44 interactions. Addition of 50 nM IGFBP-3 reduced cell viability to a comparable extent as that found using the anti-CD44 antibody (Figure 9). Prior treatment with the anti-CD44 5F12 antibodies, however, abrogated the cytotoxic effects of IGFBP3 on cell viability. Treatment with a combination of the anti-CD44 antibody and IGFBP-3 did not result in an additive negative effect on cell viability, suggesting that IGFBP-3 exerts its cytotoxic effects on A549 cell survival through a mechanism that depends on HA-CD44 interactions. Addition of either 50 nM or 150 nM humanin, in the absence of IGFBP-3, had no effect on cell survival, however, it reversed the observed cytotoxic effects of IGFBP-3 when co-added to the cells with the protein. While addition of 50 nM humanin partially reduced the inhibitory effects of IGFBP-3, a much more pronounced effect was observed when 150 nM humanin was added reversing the cytotoxicity to levels comparable to those of untreated cells. Since the 50 nM concentrations used were close to those calculated for the secreted IGFBP-3 and peptide, 48 hours post serum starvation, an interpretation of these results might be that at these concentrations, humanin is unable to fully block the cytotoxic effects of IGFBP-3. This protective effect by humanin was abolished when HA-CD44 interactions were blocked with the anti-CD44 5F12 antibodies. These results might suggest that, in the absence of prior incubation with the anti-CD44 antibodies, humanin dislodged IGFBP-3 from HA likely due to interactions with the 18residue basic amino acid sequence on IGFBP-3, a region proposed here to bind either HA or humanin, but not both simultaneously, allowing HA to re-bind its receptor, CD44, promoting cell survival. The loss of the protective effects of humanin against the cytotoxic


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effects of IGFBP-3 upon disruption of HA-CD44 interactions may point to HA-CD44 signaling as a pathway targeted by IGFBP-3.

Besides binding its known ligands, IGF-I and IGF-II, IGFBP-3 is known to have a number of other interacting partners.5,8 In serum, the IGF-IGFBP-3 complex can bind the acidlabile subunit (ALS) to form a high molecular weight heterotrimeric complex.62 ALS is known to bind residues 228–232 of IGFBP-3 which comprise part of the 18-residue basic domain implicated in this study to bind either HA or humanin. ALS is known to stabilize the IGF-IGFBP-3 complex extending the circulating half-life of IGFs.5,63,66 It may be possible to speculate that in the circulation, the humanin/glycosaminoglycans binding site is likely to be occupied by ALS, since non-complexed IGFBP-3 and IGF-1 have short circulating half-lives.

IGFBP-3–humanin–HA–CD44 may be an important hub that

regulates access of HA and/or humanin for binding to this 18-residue sequence on IGFBP-3, thereby fine tuning a regulated association/dissociation of the IGFBP3/ALS/IGF ternary complex as needed.


32

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Biochemistry


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Figure 9. Addition of humanin or blocking HA-CD44 interactions using the CD44 antibody, 5F12, markedly reduced the cytotoxic effects of IGFBP-3 in A549 cells. Viability of A549 Cells was assessed by the MTT assay. Cells were seeded in 96-well plates at 0.2 × 105 cells per well in 10% FBS-supplemented media. The following day, the cell monolayers were incubated in serum-free medium for 12 hours, then treated with either the antibodies and/or IGFBP-3 and humanin for 48 hours with the media containing the specific components in the different treatments replaced every 12h. The concentration of IGFBP-3 added was 50 nM. The concentration of humanin used was 50 nM (group labelled 1) or 150 nM (group labelled 2). Control mIgG (5 μg/mL) along with 5 μg/mL CD44 antibody (5F12) were added 2 hours prior to addition of IGFBP-3 and/or humanin. Optical density measurements (570 nm) were normalized by expressing each point in relation to the untreated control (set to 100%). Each column represents the mean ± S.D. of three independent experiments.


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CONCLUSION In this study, we characterized the binding affinities of the IGFBP-3 protein and its peptide (215KKGFYKKKQCRPSKGRKR232) in the C-terminal region of the protein, to HA and to humanin and found that the binding affinity is weaker for HA than for humanin. HA was able to effectively compete with the binding of IGFBP-3 and peptide to humanin suggesting that residues 215-232 comprise the primary region on IGFBP-3 that binds either HA or humanin, but not simultaneously. IGFBP-3 and humanin in media of A549 cells grown 48 hours post serum starvation can immunoprecipitate in a complex. However, despite the finding that IGFBP-3 and humanin interact, IGFBP-3 in the media that is able to bind HA was not complexed with humanin. This supports our conclusion that HA binding to the 215-232 segment renders it no longer accessible for binding to humanin. We also found that while CD44 is unable to compete with humanin binding to either IGFBP-3 or peptide, it is able to block HA binding to IGFBP-3. Moreover, while both IGFBP-3 and CD44 can interact with HA, humanin was effective at binding to IGFBP3, thereby enabling access of CD44 to HA. We also show that blocking HA-CD44 interactions reduced cell viability to the same extent as that found by the addition of only IGFBP-3. However, the negative effects of both treatments were not additive indicating that IGFBP-3 may reduce cell viability by disrupting HA-CD44 interactions. Addition of humanin blocked the negative effects of IGFBP-3 presumably due to competing with HA for binding to IGFBP-3. Although it is widely accepted that HA-receptor interaction is necessary for HA-induced effects, much remains to be unveiled about its basic operative mechanisms. While interaction of IGFBP-3 with humanin is well-studied and so is that of HA with its receptor,


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CD44, the interplay between these interactions is not known. Here, we investigated the binding affinities and interactions between IGFBP-3, humanin, HA, and CD44. Our results provide insights into these interactions and the molecular mechanisms underlying fundamental protein-peptide-carbohydrate interactions with possible leads to drug development and therapeutic interventions. Perturbing HMW-HA-CD44 signaling by, for example, the use of soluble CD44 or monoclonal CD44 antibodies, has been shown25,27,31,64,67–72 to inhibit cell survival while increased levels of HMW-HA promote cell growth. Thus, it is possible that binding of IGFBP-3 to HA to switch off HA-CD44 signaling while switching it on with humanin is a common biological mechanism that normally provides a delicate balance in regulating cell survival.

Our laboratory is currently

investigating this hypothesis by probing into the coordinated interactions of IGFBP-3 and humanin and consequent regulation of HA-CD44 signaling in the extracellular compartment in cell lines that express different levels of IGFBP-3 and CD44.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: (734) 487-1425. Fax: (734) 487-1496. Notes The authors declare no competing ﬁnancial interest.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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ACKNOWLEDGMENTS The authors gratefully thank Dr. Ruth Ann Armitage for the mass spectral data and Dr. David Evans for reading the manuscript. This work was supported by an Eastern Michigan University Provost Research Support Award/Chemistry Seller’s Fund.

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Interaction of Insulin-Like Growth Factor-Binding protein 3 With

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