Identification of Glycosylated Sites in Rapana Hemocyanin by Mass

Bioconjugate Chem. 2009, 20, 1315–1322

1315

Identification of Glycosylated Sites in Rapana Hemocyanin by Mass Spectrometry and Gene Sequence, and Their Antiviral Effect Pavlina Dolashka-Angelova,*,† Bernhard Lieb,‡ Ludmila Velkova,† Nina Heilen,§ Koen Sandra,| Lubomira Nikolaeva-Glomb,⊥ Aleksander Dolashki,# Angel S. Galabov,⊥ Jozef Van Beeumen,| Stefan Stevanovic,§ Wolfgang Voelter,# and Bart Devreese| Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 9 G. Bonchev St., Sofia 1113, Bulgaria, Institute of Zoology, University of Mainz, Mu¨llerweg 6, Mainz 55099, Germany, Department of Immunology, Institute for Cell Biology, University of Tu¨bingen, Auf der Morgenstelle 15, D-72076 Tu¨bingen, Germany, Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, KL Ledeganckstraat 35, 9000 Ghent, Belgium, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 G. Bonchev St., Sofia 1113, Bulgaria, and Institute of Biochemistry, University of Tu¨bingen, Hoppe-Seyler-Strasse 4, D-72076 Tu¨bingen, Germany. Received January 28, 2009; Revised Manuscript Received May 6, 2009

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana Venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

INTRODUCTION Hemocyanins (Hcs) are large, oligomeric blue coppercontaining glycoproteins that play a role as dioxygen carriers in the hemolymph of most molluscs and several arthropods (1-4). Despite their common function as dioxygen binding proteins, arthropod and molluscan hemocyanins evolved independently (3). The organization and size of molluscan and arthropodan Hc molecules differ significantly in size and oligosaccharide moieties (5-7). Carbohydrate structures of hemocyanins from Helix pomatia and Sepia officinalis and keyhole limpet hemocyanin (KLH) have been identified using different methods (8, 9). Thirteen potential N-glycosylation sites were identified in the gene sequence analysis of one subunit of Haliotis tuberculata hemocyanin (HtH1) (10) and seven in Nautilus hemocyanin FUs (11). Seven potential N-glycosylation sites are present in Octopus dofleini hemocyanin (Od) (12). A single N-linkage site and three O-linkage sites were found in the arthropod Carcinus aestuarii hemocyanin (CaH) (13, 14). Although it is generally * Corresponding author. Assoc. Prof. Dr. P. Dolashka-Angelova, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, str. G. Bonchev 9, 1113 Sofia. Tel.: 35929606163, Fax: 35928700225, E-mail: [email protected]. † Institute of Organic Chemistry, Bulgarian Academy of Sciences. ‡ University of Mainz. § Institute for Cell Biology, University of Tu¨bingen. | Ghent University. ⊥ The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences. # Institute of Biochemistry, University of Tu¨bingen.

accepted that the oligosaccharide constituents of Hc are of prime significance for their antigenicity and biomedical properties, knowledge on the carbohydrate structures of these glycoproteins is still incomplete. In this work, we focus on the structure and properties of Rapana Venosa hemocyanin, isolated from the Black Sea. Eight functional units (FUs) with molecular mass of 45-50 kDa, namely, from RvH-a to RvH-h, have been isolated from both structural subunits RvH1 and RvH2 (5, 7, 15), but the oligosaccharide moieties of only a few of them have been reported (16-19). Recently, it was shown that KLH also possesses potential antitumor activity, and this effect was ascribed to the presence of the disaccharide epitope Gal (β1-3)GalNAc, because it is crossreactive with an equivalent surface epitope of superficial bladder tumor cells (20). The KLH dependent cumulative humoral and cellular immune response is expressed as a cytolytic reduction of tumor growth. Further analyses of KLH1 and KLH2 confirmed the multiepitopic potential of these molecules (21). Hcs from other gastropods were considered to be possible substitutes for KLH as immunostimulants. It was found that marine Haliotis tuberculata (HtH) (21), Chilean gastropod, Concholepas concholepas (CcH) (22), and Rapana Venosa (RvH) (23, 24) Hcs have significant antitumor activity against mouse bladder carcinoma cells. The occurrence of xylose in Helix pomatia Hc (HpH) is considered to be highly immunogenic in mammalian species (8, 25). Subsequently, it was found that the arthropodan Penaeus monodon hemocyanin has antiviral activity against the spot syndrome virus (WSSV) and Singapore grouper iridovirus (SGIV) (26), although the antiviral mechanism is still unclear. The antiviral effect of molluscan Hcs has not been studied yet.

10.1021/bc900034k CCC: $40.75  2009 American Chemical Society Published on Web 06/08/2009

1316 Bioconjugate Chem., Vol. 20, No. 7, 2009

Since the biological effects of hemocyanins on immunostimulatory properties are attributed to their high carbohydrate content and specific monosaccharide composition, we hypothesized that antiviral effects might have a similar origin. We therefore characterized several glycopeptides from R. Venosa hemocyanin by LC/ESI-MS/MS and investigated their antiviral effects.

MATERIALS AND METHODS Materials. Rapana Venosa Hc was isolated from marine snails living in the Black Sea as described before (4). Two structural subunits RvH1 and RvH2 were separated on a Resource Q column using an FPLC system. Two functional units RvH2-b and RvH2-c were isolated from structural subunit RvH2 as described by Dolashka et al. (7, 15). All other (bio)chemicals, unless noted, were purchased from Sigma-Aldrich. RNA Extraction and RT-PCR. RNA was extracted using the RNeasy extraction-Kit (Qiagen) according to the manufacturer’s instructions using 200 mg of foot tissue. RT-PCR was performed using 1 µg of RNA, Transcriptor reverse transcriptase (Roche), and a degenerated hemocyanin specific oligonucleotide (27). PCR was performed using 5 µL of the RT assay. Each PCR was supplied with 1 µM primer, 200 µM dNTP, 3 mM MgCl2, 5 µL of 10× PCR buffer, and 2.5 units of recombinant Taq polymerase (Invitrogen, Germany). Samples were denatured for 2 min at 94 °C, followed by 35 amplification cycles (10 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C) and final extension for 10 min at 72 °C. Gel Electrophoresis, DNA Purification, and Sequencing Reactions. PCR products were separated by electrophoresis in a 0.8% agarose gel (1× TBE buffer) and extracted using the Peqlab gel extraction kit (Peqlab, Germany). The isolated fragments were either sequenced directly or after being cloned using the StrataClone PCR Cloning Kit (Startagene, USA). Sequencing reactions were performed with the Taq DyeTerminator system (Perkin-Elmer) using standard primers and electrophoretically analyzed by Genterprise (Germany). Bioinformatics. Alignments were created by ClustalX V1.81 (28) and optimized manually using Genedoc (29). Database searches and further analyses were performed using sequence analysis tools [e.g., Blastn and Blastx (30) available at the NCBI (www.ncbi.nlm.nih.gov). 3D models were created using the Swiss PDB viewer (31), and phylogenetic analyses were carried out using Mega 3.1 (32). Mass Spectrometric Analysis of Glycopeptides by Nanoelectrospray MS/MS. Four milligrams of RvH1 and RvH2 were digested with trypsin, and the glycopeptides were isolated as described by Dolashka-Angelova et al. (16). HPLC fractions, detected at a wavelength of λ ) 206 nm, were collected, lyophilized, and analyzed for carbohydrates with orcinol/H2SO4 on silica gel plates (18). Glycopeptides containing fractions were analyzed by nanoelectrospray ionization (ESI) mass spectrometry (MS) on a hybrid quadrupole orthogonal acceleration timeof-flight tandem mass spectrometer (Q-TOF; Micromass, Manchester, United Kingdom) as described (33). Alternatively, glycopeptides were sequenced on a Q-Trap LC/MS/MS system (Applied Biosystems/MDS Sciex, Concord, ON, Canada) equipped with a nanospray ion source (Protana, Odense, Denmark) as described by Sandra et al. (19). Antiviral Effects of the Glycosylated and Nonglycosylated Functional Units. The in Vitro hemocyanin antiviral effects on the replication of poliovirus type 1 (LSc-2ab), coxsackievirus B1 (CV-B1), and respiratory syncytial virus (RSV) were studied using native RvH and its glycosylated (RvH-c) and nonglycosylated (RvH-b) functional units at a concentration of 1000 µg/mL in 15 mM phosphate buffer, pH 7.1. Polio virus type 1 (LSc-2ab) and CV-B1 were grown in FL cells and RSV was grown in Hep-2 cell cultures. The cultures were grown in

Dolashka-Angelova et al.

a growth medium of DMEM (Gibco BRL, USA), containing 5% fetal calf serum, supplemented with antibiotics (penicillin 100 IU/mL, streptomycin 100 µg/mL, and gentamycin 50 µg/ mL) and 20 mM HEPES buffer (Gibko BRL, USA) in a humidified atmosphere at 37 °C and 5% CO2. Cells were routinely subcultured twice a week. Viruses were replicated on monolayer cells in a maintenance medium of DMEM, supplemented with 0.5% fetal calf serum, 20 mM HEPES buffer, and antibiotics. Cytotoxicity Test. For cytotoxicity tests, Hep-2 cells were seeded in flat-bottomed 96-well plates by adding 0.2 mL per well of the cell suspension, containing (1.5-2) × 105 cells/ mL. After formation of a cell monolayer, growth medium was discarded and 0.2 mL containing 0.5 µg serial dilution concentrations of the tested substances, diluted in a maintenance medium, were added, followed by further incubation of cells in a humidified atmosphere at 37 °C and 5% CO2 and monitoring the microscopic cytotoxic effect after 24 and 48 h. The highest concentration showing no cytotoxic effect was recorded at 48 h post-inoculation and was considered as the maximum of tolerated nontoxic concentration (MTC). Antiviral Test. The cytopathic effect (CPE) inhibition test was used to measure the antiviral effect of the compounds against the replication of polio virus type 1 (LSc-2ab), CV-B1, and RSV. Briefly, monolayer cells in 96-well plates were inoculated with 0.1 mL virus suspension containing 100 or 10 CCID50. After virus adsorption for 1 h at room temperature for polio virus type 1 (LSc-2ab) and CV-B1, and for 2 h at 37 °C and 5% CO2 for RSV, excessive virus suspension was discarded, and cells were inoculated with 0.2 mL of maintenance medium containing 1 mg/mL RvH-c or RvH-b. Cells were incubated (Hera Cell, 150, Hereaus, Germany) in a humidified atmosphere at 37 °C and 5% CO2. Virus-induced CPE was scored daily by inverted light microscopy (Olympus CK40, Japan) at 125× and 400× magnification on a 0-4 basis (4 representing total cell destruction and 0 the intact cell monolayer) until the appearance of its maximum in the untreated virus control wells (with no compound in the maintenance medium). This was on the 48th hour for polio virus type and CV-B1 and on the 120th hour for RSV. Virus-induced CPE in treated and nontreated virus control wells was compared, and the percent of CPE inhibition was calculated. Virucidal Test. The quantitative suspension test was used to detect the virucidal effect of the compounds. Equal volumes of undiluted virus suspension and each of the compounds at their highest initial concentrations were mixed and kept for 1 h at room temperature. The residual infectious virus was determined in monolayer cells by inoculating 0.1 mL of serial 10fold dilutions of the virus suspension samples, followed by 1 or 2 h adsorption at 37 °C and 5% CO2. Then, excessive virus was discarded, and cells were inoculated with 0.2 mL of maintenance medium and further incubated at 37 °C and 5% CO2. The virus CPE was scored daily until its maximum in the virus control wells.

RESULTS AND DISCUSSION KLH has already been in use for several years as an immunotherapeutic agent in the treatment of bladder carcinoma and other cancer types (34). The relevance of specific glycosylation patterns as a differentiating factor for immunostimulatory properties has already been raised in studies on the hemocyanin isoforms KLH1 and KLH2 of Megathura crenulata (20, 21). Molluscan hemocyanin genes contain several potential N-glycosylation sites, and some of those have been already effectively demonstrated to be glycosylated using a series of analytical strategies (8, 13). For a functional unit of Octopus dofleini hemocyanin OdH-g, this has been confirmed by X-ray

Glycosylated Sites in Rapana Hemocyanin

Bioconjugate Chem., Vol. 20, No. 7, 2009 1317

Table 1. Amino Acid Sequence and Carbohydrate Structure of Glycopeptides Isolated from RvH and Analyzed Using Different Techniquesa glycopeptides

glycans

m/z

ESI-MS 1 2 3 4 5 6

[QK]AENLTTTR AENLTTTR HHGHV[ · · · K · · · N · · · ]R FSGEVDGHNTSR YE[IL]HAVNGST[IL]AA[IL] YE[IL]HAVNGST[IL]AA[IL]

7 8

MGQYGNLSTNNTR SVNGTLLGSQILGKPY SVNGTLLGSQILGK FSGEVDGHNTSR AENITTTR FANATSIDGPNA EMLTLNGTNLA IHSYSGSYINASLLHGPSII

Fuc Man3GlcNAc2Hex3 Fuc Man3GlcNAc2Hex3 Fuc Man3GlcNAc2Hex3 Man3GlcNAc2 Hex2Man3GlcNAc2 Hex3Man3GlcNAc2

1036 [M+2H]2+ 972 [M+2H]2+ 1396 [M+2H]2+ 1099 [M+2H]2+ 1338 [M+2H]2+ 1419 [M+2H]2+

Q trap-LC/MS/MS

9 10 11 12 13 a

Hex Man3HexNAc2 Fuc Man3GlcNAc2 Man3GlcNAc2 Man3GlcNAc2 Fuc Man3GlcNAc2 SO4 MeHexAMeHexNAc2Man3GlcNAc2 MeHex2AHexNAc2Man3GlcNAc2 MeManMan2GlcNAc2

837.97[M+2H]3+ 1099 [M+2H]2+ 972 [M+2H]2+ 2786 [M+H]+ 2846 [M+H]+ 2848 [M+H]+

The exact order of the residues between brackets could not be established, and X represents unknown amino acids.

Figure 1. Fragment spectra of the glycopeptide G1 with determined m/z 1338.16 [M+2H]2+. (a) MS/MS analysis of the sugar moiety, using a collision energy of 37 eV. Annotation of sugar fragments. The insert shows the complete carbohydrate structure and the cleavage points leading to the respective fragments. * corresponds to the doubly charged ions. (b) Pseudo-MS/MS/MS experiment (in-source and collision-induced fragmentation) of the peptide moiety (YEXHAVN*GSTXAAX) still carrying one HexNAc (m/z 1662.09, [M-H]+), using a cone voltage of 95 V and a collision energy of 80 eV. R corresponds to the peptide.

crystallography (35). Previous analyses of the oligosaccharide moiety of RvH revealed a small set of carbohydrate structures (16-19). We extended this study here implementing advanced separation and mass spectrometric techniques to analyze the binding sites and the carbohydrate heterogeneity of the structural subunits RvH1 and RvH2. We used two approaches to analyze the glycosylation sites in Rapana Venosa hemocyanin. The first approach included the

isolation of glycopeptides from structural subunits RvH1 and RvH2, and analysis of their amino acid sequences and structures of the linked oligosaccharides by mass spectrometry. Thirteen glycopeptides, given in Table 1 (No 1-13), were identified using ESI-MS and Q-Trap-LC/MS/MS. Some of them were previously characterized by capillary electrophoresis (19) or HPLC/electrospray ionization mass spectrometry (17, 18). The second approach involved identification of the linkage sites by gene sequencing.



Figure 2. Precursor (a) scan of the ion at time 31.24 min of the chromatographic separation (insert) and enhanced product ion (EPI) scan of the ion (b) at m/z 837.97.

Glycopeptides were isolated after overnight digestion of the structural subunits RvH1 and RvH2 with trypsin, and the resulting peptides were subjected to reversed phase separation using a Nucleosil 7 C18 column. Fractions testing positive in the orcinol test were lyophilized and studied by mass spectrometry. Characterization of Glycopeptides Using HPLC Fractions Analyzed by Flow Injection Analysis on a Q-TOF ESI-MS. Six glycopeptides were characterized in detail by analyzing the corresponding HPLC fraction by ESI-MS/MS. As an example, we outline here the result of a peptide (Table 1, No 5) that appeared as a doubly protonated molecule of m/z 1338.16 [M+2H]2+ in the MS spectrum (not shown). The MS/MS spectra obtained from this glycopeptide are displayed in Figure 1. Figure 1A shows the MS/ MS spectrum after collisional induced dissiocation. The fragment ion at m/z 1661.71 corresponds to the peptide (designated R-, m/z 1459.75), which is N-glycosylated, with a single residual GlcNAc residue. The spectrum is further dominated by glycan fragments, which correspond to a classical mannose type of oligosaccharide, consisting of two N-acetylhexosamines (HexNAc) monomers as well as five hexose (Hex) residues. In a pseudo-MS (3) experiment, the sugar side chain was removed by in-source fragmentation, and the peptide with one remaining HexNAc residue was further fragmented in the hexapole collision cell. This allowed the sequence analysis of the peptide and the determination of the site to which the sugar chain was attached (Figure 1B). The amino acid sequence of the peptide was revealed to be YEXHAVN*GSTXAAX. The

glycosylation site N* was identified as part of the typical Nglycosylation motif N-aa-S/T, where aa can be any amino acid except Proline. X represents a leucine or isoleucine residue, which could not be distinguished by the applied MS methods. Five additional glycopeptides (Table 1, peptides 1-6) were characterized in the same way. Characterization of Glycopeptides Using Q-Trap LC/MS/ MS System. The Q-Trap system with its capabilities to perform typical triple quadrupole scans was additionally used for this purpose. Glycopeptides, selectively detected in a proteolytic mixture by the appearance of collisionally induced marker oxonium ions such as m/z 163 (Hex+), 204 (HexNAc+), or 366 (HexHexNAc+), were sequenced. The insert of Figure 2a shows the LC/MS/MS total ion current (TIC) chromatogram of the precursor ion scan (monitoring m/z 204) of the HPLC fraction at time 31.24 min. The enhanced resolution scan (not shown) showed that the glycopeptide with mass 2511.91 eluting at this moment was triply charged at m/z 837.97 [M+3H]3+. The precursor ion scan at time 31.24 min is presented in Figure 2a. The spectrum is dominated by glycan fragmentation corresponding to the Domon/Costello nomenclature. However, peptide fragmentation (Roepstorff/Biemann cleavages) became more dominant when the collision energy was increased, allowing one to deduce the peptide sequence MGQYGD(I/L)STNNTR from the series of y- and b-ions (Figure 2b). The ion b13 (m/z 1439.6) or y13 (m/z 1457.5) corresponds to the peptide, which contains two potential linkage



Figure 3. (A) Partially primary structure of functional unit RvH-b, of RvH as predicted from its cDNA sequence. (B-insert) A multiple sequence alignment of the linker intron identified in RvH-c in comparison to Haliotis tuberculata (two isoforms: HtH1-b/c, HtH2- b/c). (B) A multiple sequence alignment of the RvH-c with other FUs-c (the N-glycosylated site in RvH-c in green, other such sites are underlined. Residues in red and blue are conserved, or nearly conserved, respectively). Abbreviations of species: RvH, Rapana Venosa Hc, AcH, Aplysia californica Hc, HtH, Haliotis tuberculata Hc. The secondary structural elements β4 and β5 are those occurring in the 3D structure of Octopus dofleini Hc (35).

sites -D(L/I)S- and -NNT-. Normally, D(L/I)S is not expected to be a linkage site, but, just in the unlikely event that deglycosylation of a glycan linked asparagine occurred during sample preparation, we thoroughly investigated this option. The ion y7 at m/z 806.4 corresponds to the C-terminal fragment of the peptide -(I/L)STNNTR, and the ion at m/z 1009.6 represents the same fragment still containing one GlcNAc moiety. This suggests that only the linkage site -NNT- is glycosylated, most likely via a high mannose-like structure. The ion at m/z 1660.9 corresponds to the intact peptide, represented as ion y13 (m/z 1457.5), which is N-glycosylated with a single GlcNAc residue. The glycan structure of this peptide could be revealed by this MS/MS spectrum, which displayed the typical ions (represented as Y and B) at m/z 204.1 (GlcNAc), m/z 366.2 (HexGlcNAc), m/z 528.3 (Hex2GlcNAc), 690.5 (Hex3GlcNAc), and 852.4 (Hex4GlcNAc) (Figure 2B). Combining all data, the carbohydrate structure of 1054.0 Da (Hex2Man3GlcNAc2) could be suggested. Additional carbohydrate structures were analyzed in the same way and authentic binding sites could be revealed. The results are summarized in Table 1. Identification of the Gene Sequence of Two RvH Functional Units. To better frame these results, we sequenced the cDNA of two FUs from RvH. To obtain the complete coding sequence for R. Venosa hemocyanin (RvH) subunits, we performed RTPCR using degenerated primers derived from the copper binding site A of H. tuberculata hemocyanin (36). The obtained fragments were cloned, sequenced, and identified by Blast analysis. We were able to determine the sequence of one

hemocyanin coding fragment of 1668 bp in length coding for 556 amino acids, which in fact consisted of two subfragments. Since single functional units (FU) of hemocyanins can be assigned to their orthologs by multiple sequence alignment (27), we assume these two fragments partially code for of FU-b (Figure 3A) and FU-c (Figure 3B). The fragment of RvH-b comprises 316 amino acids lacking the first ca. 100 amino acids referring to the complete FU-b of known hemocyanins (10, 27, 36). The fragment of the FU-c comprises 240 amino acids, lacking ca. 200 residues of the C-terminus of complete FU-c. However, we can identify 14 amino acids to be part of a conserved phase 1 linker-intron, which exactly corresponds to both the intron phase and the position of the linker introns of functional unit b or c from the first structural subunit of H. tuberculatos (HtH1-b/c) and functional unit b or c from the second structural subunit of H. tuberculatos (HtH2-b/c) (Figure 3B-insert). The partitioning of the complete fragment into two separate FUs was done according to the known gene structures of H. tuberculatos, O. dofleini, and N. pompilius hemocyanins (10-12, 36) (Figure 3A,B). Concerning the intron in the gene region of RvH-bc, we should mention that two types of introns were recognized in each gene of Haliotis tuberculata hemocyanin and Octopus dofleini hemocyanin: (i) introns positioned within the linker coding region between two functional units, referred to as “linker introns”, and (ii) introns within a functional unit exon, consequently termed “internal introns”. All hemocyanin linker introns identified so far are strictly phase 1 introns. According to these findings, the intron we observed in RvH is such a linker intron.


Figure 4. (A) Matrix of percentage identity between known hemocyanin sequences of FUs from Rapana Venosa and Haliotis tuberculata. (B) A radial phylogenetic tree with of molluscan hemocyanin functional units sequences from three FUs from Rapana Venosa (RvH-a,c,e) and Haliotis tuberculata (two isoforms: HtH1, HtH2).

In addition to the assignment of the orthologous FUs, the multiple sequence alignment of the separated FUs shows that RvH-b shares an identity of 52% and 51% with the vetigastropod hemocyanin isoforms HtH1-b and HtH2-b of Haliotis tuberculata, respectively, whereas the corresponding FU-c shares an identity of 57% and 60% with HtH1-c and HtH2-c, respectively (Figure 4A). This clearly suggests a closer relationship of RvH to HtH, which was also suggested by biochemical studies based on immunological assays. Due to the similar identity relationships of HtH1 and HtH2, the closer relationship of RvH and HtH2 cannot be seen in the molecular tree (Figure 4B). Moreover, they do not differ notably from any typical hemocyanin primary structure (Figure 4B) (27). No significant similarity to known N-terminal sequence data of isolated RvH1-b/c or RvH2-b/c subunits could be detected (16). Comparisons of RvH-c and the deduced complete primary structures of other molluscan FUs-c shown in Figure 3C (only partial alignments are shown), reveal that only four of the nine c-FUs of Rapana Venosa (RvH-c), H. tuberculatos (HtH2-c), O. dofleini (OdH-c), and N. pompilius (NpH-c) contain putative N-linkage sites. Localization of the Carbohydrate Linkage Sites from the Gene Sequence. No carbohydrate linkage site was identified within the 316 amino acids of RvH-b. The finding that the orcinol/H2SO4 glycosylation test was negative for FU-b (data not shown) is thus corroborated by the gene sequence data and suggests that the N-terminal region of 100 residues contains no N-glycosylation site. Both isoforms of HtH-b contain at least one potential N-glycosylation site, which cannot be detected within the C-terminal 316 residues of RvH-b. The finding that RvH-c does react with the reagent likewise corresponds to the gene sequence data, which reveal the presence


Figure 5. Precursor ion scan (A) at time 30.71 min of the chromatographic separation (insert) and enhanced product ion (EPI) scan of the ion (B) at m/z 1099.

of a glycosylation site at position 143. This NTS site is unique compared to the other sequences given in Figure 3C and matches perfectly well with the site identified the glycopeptide of mass 1099 [M+2H]2+ by mass spectrometry (Table 1, peptides 4 and 9). Several mass spectral results revealed that the carbohydrate structure Man3GlcNAc2 is branched to the RvH-c peptide FSGEVDGHNTSR. The precursor ion scan (monitoring m/z 204) at time 30.71 min during chromatography, the enhanced resolution (ER) scan of the most dominant ion (973), and the MS/MS spectrum of that ion are presented in Figure 5. It was determined that the glycopeptide has a 2× charge. By subtracting the peptide masses from the molecular ion 1099.19 [M+2H]2+ ([1099.19.2 × 2] - 1 ) 2197.38), the glycan structure could be derived. The Man3GlcNAc2 glycan appears to occupy the -NTS- glycosylation site. This was confirmed by further investigation of the MS/MS spectrum. The ions at m/z 680.1 and 1508.5 correspond to the ion y4 (-NTSR-) and y11 (peptide), attached with one GlcNAc. The ion y11 at m/z 1305.6 corresponds to the peptide without the glycan. The m/z values for y8 and y9 differs by the mass of one tryptophan residue, and the peptide sequence FSWVDGHNTSR was determined. However, it differs by the gene sequence, because mass spectrometric fragmenting around a Gly residue often results in very small peaks and because a glycine-glutamic acid dipeptide is isobaric to a single tryptophan. Therefore, we now conclude that the gene sequence is likely to be correct. The position, determined by model building, shows that the carbohydrate chain is exposed at the surface of the functional unit (Figure 6), at a similar position within the well-characterized structure of cephalopod Octopus dofleini (OdH) hemocyanin (35). In addition, it might be essential for establishing or maintaining any contact with other FUs and might contribute to the overall stability of the oligomeric hemocyanin; this remains to be determined.



(CPE) inhibition test was used to measure the antiviral effect of hemocyanin against the replication of poliovirus type 1 (LSc2ab), CV-B1, and RSV. The results showed no antiviral effects of native RvH and of both FUs against polio virus type 1 (LSc2ab) and CV-B1 (data not shown). However, the results presented in Table 2 show that the glycosylated FU RvH-c possesses some antiviral effect against the replication of RSV, at both viral doses tested. When the effect was tested against the lower virus dose of 10 CCID50, the antiviral activity reached even 100% inhibition of the virus-induced cytopathic effect. Although not so strongly expressed, the antiviral effect is evident also at higher virus doses of 100 CCID50 (which is the standard virus dose used in the CPE inhibition test), exceeding the 50% inhibition of the virus CPE. In contrast, non-glycosylated FU RvH-b does not reveal any antiviral activity against the replication of RSV. The compounds also do not show antiviral effect against the replication of poliovirus type 1 (LSc-2ab) and CV-B1. In conclusion, CPE-inhibition tests of the native RvH and both FUs against the replication of polio virus type 1 (LSc2ab), CV-B1, and RSV revealed that there is no inhibitory effect on these viruses for native RvH and for non-glycosylated FU-b. However, the glycosylated FU RvH-c does show antiviral effect in the case of RSV replicated in HEp-2 cells. In order to understand the mechanism of this effect, further investigations are under way. Figure 6. Presumed 3D structure of FU RvH-c, based on the strongly homologous structure of Octopus dofleini Hc (35) and position of carbohydrate structure attached to the polypeptide chain. Table 2. Testing for Antiviral Activity against the Replication in Vitro of RSV CPE score on a 0-4 basis (mean of four wells) 100 CPE 10 CPE concentration CCID50/ inhibition CCID50/ inhibition compounds (µg/mL) well % well % RvH RvH-b RvH-c

1000 0 1000 0 1000 0

3.5 3.5 3.5 3.5 1 3.5

0 0 71.4 -

1.5 1.5 1.5 1.5 0 1.5

0 0 100 -

Antiviral Effect of RvH. Molluscan hemocyanins possess certain inhibitory properties against bacteria, but, so far, it was not shown to have any antiviral potential. We demonstrate here, for the first time, the inhibitory effect of molluscan hemocyanin RvH against viruses. We investigated whether this inhibition effect is linked to the presence of carbohydrate chains in RvH. To study the antiviral effect of RvH-oligosaccharides, polio virus type 1 (LSc-2ab), coxsackie virus B1 (CV-B1), and respiratory syncytial virus (RSV) were treated with native RvH and with nonglycosylated and glycosylated functional units RvH-b and RvH-c (0.45 and 2.0 mg/mL). Cytotoxicity, virucidal, and antiviral effects of glycosylated RvH-c and nonglycosylated FUs RvH-b were studied in FL or HEp-2 monolayer cells depending on the virus tested. The compounds were nontoxic to monolayer cells even at the highest initial levels of concentration. Monolayer cells, treated with the highest given concentrations of the tested substances, remained completely intact, and no visible changes were detected by inverted light microscopy 24 and 48 h after treatment. Cytotoxicity control was carried out in parallel with the CPE inhibition test. Cells in this case were monitored for signs of toxicity until 120 h p.i. No toxicity was recorded either. Virucidal and Antiviral Tests. The virucidal quantitative suspension test did not show any virucidal effect against polio virus type 1 (LSc-2ab), CV-B1, and RSV. The cytopathic effect

ACKNOWLEDGMENT This work was supported by a research grant by the Ministry of Sciences and Education through the grant: DAAD-17/2007 (VU-L-310/07) and TK01/496, DFG (Deutsche Forschungsgemeinschaft) (Germany), Fund for Scientific Research-Flanders (FWO-Vlaanderen) through project VS.011.06N and CNR (Italy). Assoc. Prof. P. Dolashka-Angelova also thanks DAAD for granting a scholarship.

LITERATURE CITED (1) Paoli, M., Giomi, F., Hellmann, N., Jaenicke, E., Decker, H., Di Muro, P., and Beltramini, M. (2007) The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4 × 6-meric protein of Upogebia pusilla (Crustacea). Gene 398, 177–182. (2) Van Holde, K. E., Miller, K. I., and Decker, H. (2001) Hemocyanins and invertebrate evolution. J. Biol. Chem. 276, 15563–15566. (3) Burmester, T. (2001) Molecular evolution of the arthropod hemocyanin superfamily. Mol. Biol. EVol. 18, 184–195. (4) Dolashka-Angelova, P., Schwarz, H., Dolashki, A., Stevanovic, S., Fecker, M., Saeed, M., and Voelter, W. (2003) Oligomeric stability of Rapana Venosa hemocyanin (RvH) and its structural subunits. Biochim. Biophys. Acta 1646, 77–85. (5) Dolashka, P., Genov, N., Pervanova, K., Voelter, W., Geiger, M., and Stoeva, S. (1996) Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits. Biochem. J. 315, 139–144. (6) Giomi, F., and Beltramini, M. (2007) The molecular heterogeneity of hemocyanin: Its role to the adaptive plasticity of Crustacea. Gene 398, 192–201. (7) Dolashka-Angelova, P., Stevanovic, S., Dolashki, A., Devreese, B., Tzvetkova, B., Voelter, W., Van Beeumen, J., and Salvato, B. (2007) A challenging insight on the structural unit 1 of molluscan Rapana Venosa hemocyanin. Arch. Biochem. Biophys. 459, 50–58. (8) Gielens, C., De Geest, N., Compernolle, F., and Preaux, G. (2004) Glycosylation sites of hemocyanins of Helix pomatia and Sepia officinalis. Micron 35, 99–100.

1322 Bioconjugate Chem., Vol. 20, No. 7, 2009 (9) Wuhrer, M., Robijn, M. L. M., Koeleman, C. A. M., Balog, C. I. A., Geyer, R., Deelder, A. M., and Hokke, C. H. (2004) A novel Gal(β1-4)Gal(β1-4)Fuc(R1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet hemocyanin (KLH) N-glycans. Biochem. J. 378, 625–632. (10) Lieb, B., Altenhein, B., and Markl, J. (2000) The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata). J. Biol. Chem. 25, 5675–81. (11) Bergmann, S., Lieb, B., Ruth, P., and Markl, J. (2006) The hemocyanin from a living fossil, the cephalopod Nautilus pompilius: protein structure, gene organization, and evolution. J. Mol. EVol. 62, 3362–74. (12) Miller, K. I., Cuff, M. E., Lang, W. F., Varga-Weisz, P., Field, K. G., and van Holde, K. E. (1998) Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications. J. Mol. Biol. 278, 827–842. (13) Dolashka-Angelova, P., Beltramini, M., Dolashki, A., Salvato, B., and Voelter, W. (2001) Carbohydrate composition of Carcinus aestuarii hemocanin. Arch. Biochem. Biophys. 389, 153– 158. (14) Dolashka-Angelova, P., Dolashki, A., Savvides, S. N., Hristova, R., Van Beeumen, J., Voelter, W., Devreese, B., Weser, U., Di Muro, P., Salvato, B., and Stevanovic, S. (2005) Structure of hemocyanin subunit CaeSS2 of the crustacean mediterranean crab Carcinus aestuarii. J. Biochemistry 138, 303–312. (15) Stoeva, S., Dolashka, P., Pervanova, K., Genov, N., and Voelter, W. (1997) Multidomain structure of the Rapana thomasiana (Gastropod) hemocyanin structural subunit RHSS1. Comp. Biochem. Physiol. 118 B, 927–934. (16) Dolashka-Angelova, P., Beck, A., Dolashki, A., Stevanovic, S., Beltramini, M., Salvato, B., Hristova, R., Velkova, L., and Voelter, W. (2004) Carbohydrate moieties of molluscan Rapana Venosa hemocyanin. Micron 35, 101–104. (17) Dolashka-Angelova, P., Beck, A., Dolashki, A., Beltramini, M., Stevanovic, S., Salvato, B., and Veolter, W. (2003) Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana Venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion. Biochem. J. 374, 185–192. (18) Beck, A., Hillen, N., Dolashki, A., Stevanovic, S., Salvato, B., Voelter, W., and Dolashka-Angelova, P. (2007) Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry. Biochimie 89, 938–949. (19) Sandra, K., Dolashka-Angelova, P., Devreese, B., and Van Beeumen, J. (2007) Novel methodologies in glyco-analysis reveal new insights in Rapana Venosa hemocyanin N-glycosylation. Glycobiology 17, 141–56. (20) Wirguin, I., Suturkova Milosevic, L., Briani, C., and Latov, N. (1995) Keyhole limpet hemocyanin contains Gal(β1-3)GalNAc determinants that are cross-reactive with the T antigen. Cancer Immunol. 40, 307–310. (21) Markl, J., Lieb, B., Gebauer, W., Altenhein, B., Meissner, U., and Harris, R. (2001) Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH. J. Cancer Res. Clin. Oncol. 127 (2), R3–R9. (22) (a) Moltedo, B., Faunes, F., Haussmann, D., De Ioannes, P., De Ioannes, A. E., Puente, J., and Becker, M. I. (2006)

Dolashka-Angelova et al. Immunotherapeutic effect of Concholepas hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among hemocyanins. J Urol. 176, 2690–2695. (23) Toshkova, R., Velkova, L., Voelther, W., and DolashkaAngelova, P. (2006) Protective effect of Rapana Venosa hemocyanin (RvH) on survivability of hamsters with transplanted myeloid graffi tumours. C. R. Acad. Bulg. Sci. 59, 977–982. (24) Toshkova, R., Ivanova, E., Nastke, M., Stevanovic, S., Velkova, L., Voelter, W., and Dolashka-Angelova, P. (2006) Hemocyanins as immunostimulators. Global J. Mol. Sci. 1, 22– 32. (25) Van Kuik, J. A., Sijbesma, R. P., Kamerling, J. P., Vliegenthart, J. F. G., and Wood, E. J. (1987) Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis. 3-O-MethylD-galactose and N-acetyl-D-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein. Eur. J. Biochem. 169, 399–411. (26) Zhang, X., Huang, C., and Qin, Q. (2004) Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon. AntiViral Res. 61, 93–99. (27) Lieb, B., Altenhein, B., Markl, J., Boisgue´rin, V., Gebauer, W., and Markl, J. (2004) cDNA sequence, protein structure, and evolution of the single hemocyanin from Aplysia californica, an opisthobranch gastropod. J. Mol. EVol. 59, 536–45. (28) Topham, R., Tesh, S., Westcott, A., Cole, G., Mercatante, D., Kaufman, G., and Bonaventura, C. (1999) Disulfide bond reduction: A powerful, chemical probe for the study of structurefunction relationships in the hemocyanins. Arch. Biochem. Biophys. 369, 261–266. (29) Nicholas, K. B., and Nicholas, H. B., Jr. (1997)GeneDoc: analysis and. visualization of genetic variation. http://www. psc.edu/biomed/genedoc/. (30) Bairoch, A., and Apweiler, R. (1999) The SWISS-PROT protein sequence data bank and its supplement TrEMBL. Nucleic Acids Res. 27, 49–54. (31) Guex, N., and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723. (32) Tamura, T., and Akutsu, T. (2007) Subcellular location prediction of proteins using support vector machines with alignment of block sequences utilizing amino acid composition. BMC Bioinformatics 8, 466. (33) Carr, S. A., Huddleston, M. J., and Bean, M. F. (1993) Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry. Protein Sci. 2, 183–196. (34) Lamm, L. D., Petegolli, I. J., and Riggs, R. D. (2000) Keyhole limpet Hemocyanin immunoterapia del cancero alla vescica: laboratorio e studi clinici. Eur. Urol. 37, 41–44. (35) Cuff, M. E., Miller, K. I., van Holde, K. E., and Hendrickson, W. A. (1998) Crystal structure of a functional unit from Octopus hemocyanin. J. Mol. Biol. 278, 855–870. (36) Altenhein, B., Markl, J., and Lieb, B. (2002) Gene structure and hemocyanin isoform HtH2 from the mollusc Haliotis tuberculata indicate early and late intron hot spots. Gene 301, 53–60. BC900034K

Identification of Glycosylated Sites in Rapana Hemocyanin by Mass

Recommend Documents