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Originally published In Press as doi:10.1074/mcp.M500324-MCP200 on October 31, 2005.
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Molecular & Cellular Proteomics 5:226-233, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Elucidation of N-Glycosylation Sites on Human Platelet Proteins

A Glycoproteomic Approach*

Urs Lewandrowski{ddagger}, Jan Moebius{ddagger}, Ulrich Walter§ and Albert Sickmann{ddagger},

From the {ddagger} Protein Mass Spectrometry and Functional Proteomics Group, Rudolf Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany and the § Department of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Josef-Schneider Str. 2, 97080 Wuerzburg, Germany


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

During recent years substantial advances have been made within and by the field of proteomics. In addition to the common differential proteomic analyses focusing on the characterization of protein patterns in two different, well controlled states, e.g. healthy and diseased, the inventory of proteins from complete organisms, cell types (1), or organelles (2) received considerable attention. However, most of these investigations paid little attention to the post-translational modifications of the proteins investigated, e.g. glycosylation and phosphorylation sites. Most data in this respect are still obtained by studies of individual proteins.

Glycosylations are known as a most heterogenic group of modifications to proteins in general. Abundant types of O- and N-glycosylation comprise most commonly serine, threonine, and asparagine residues as well as glycosylphosphatidylinositol anchors. Furthermore, carbohydrate additions such as C-mannosylation and O-fucosylation are known to be present on proteins of interest (3). The enormous complexity of those modifications often hampers a detailed analysis due to low sample amounts of individual isoforms and structural diversity. O-Glycosylations for instance may be distributed to eight different core structures with numerous possible extensions and modifications (4). Structural glycan databases comprise over 2000 different sugar modifications described so far on proteins (5).

However, even basic data upon glycosylation of platelet proteins are still rare despite the importance of glycosylations e.g. in cell-cell recognition (6), ligand binding, antigen presentation, pathogen binding, and their role in disease. The site of carbohydrate attachment to the peptide backbone is of primary interest. Furthermore, the detailed structure of the attached glycan and the distribution of isoforms over a range of glycosylation sites is a potential research topic. For N-glycosylation the search for glycosylation sites is supported by a known consensus sequence (NX(S/T) where X is not equal to Pro). In combination e.g. with structural data derived from crystallization studies and experimental details regarding the type of glycosylation, this information will aid in confirmation of previous findings, e.g. protein-glycan interaction studies. The presence of modification sites on interaction surfaces may reveal completely new features in this context.

For elucidation of N-glycosylation sites a very convenient method makes use of the properties of PNGaseF1 (7). This enzyme not only cleaves all types of N-glycans, with few exceptions (8), from the polypeptide backbone but also possesses additional amidase activity during this process. Therefore, PNGaseF converts asparagine to aspartic acid during the cleavage reaction. This results in a 1-Da mass shift detectable by most of the recently available mass spectrometers. A major drawback regarding analysis is the high number of non-glycosylated peptides in a complete cell digest. Usually only the main structural protein compounds, e.g. actin and filamin, are identified rather than low abundance molecules, which are of superior interest. For specific enrichment of glycopeptides two methods are used. Because mainly N-glycosylated proteins are targeted in this approach we use concanavalin A for the trapping of a broad range of N-glycans (9, 10). A 2-fold lectin affinity chromatography yields mainly glycopeptides as the dominant species. Thereby, trapped glycoproteins are eluted from the lectin column and digested with trypsin. After removal of non-glycosylated peptides by a second lectin affinity chromatography a derivatization is performed using PNGaseF. A less specific approach, not limited to certain types of N-glycans, is included in the study by use of chemical derivatization of the glycan residues and trapping of glycosylated proteins on hydrazide-functionalized resins. Thereby, reactive aldehyde groups are introduced into the sugar side chains by periodic acid oxidation of vicinal diols present in most hexoses. Those aldehydes can be covalently coupled to hydrazide-derivatized agarose beads. Elution of glycopeptides is performed by use of PNGaseF. Due to the complexity of the resulting peptide mixtures a separation prior to mass spectrometry is necessary. We used nano-reversed-phase HPLC in combination with ESI-MS/MS. The instruments used combine high sensitivity and selectivity as well as the necessary resolution and mass accuracy for the analysis of derivatized glycopeptides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—
Agarose-bound concanavalin A was purchased from Vector Laboratories, Burlingame, CA, and hydrazide beads were from Perbio Science, Bonn, Germany. PNGaseF was obtained from Roche Applied Science. Trypsin was ordered from Fluka, Steinheim, Germany. All other chemicals used in this study were ordered from Sigma and were of analytical or higher grade.

Purification of Human Platelets—
Purified human platelets were prepared using modifications of published procedures (11) including repeated centrifugation and washing steps and apheresis-derived platelet preparations (Department of Transfusion Medicine, University of Wuerzburg, Germany). To remove plasma proteins and potentially contaminating leukocytes and erythrocytes the samples were centrifuged twice in 50-ml centrifugation tubes at 380 x g for 15 min at room temperature. Both pellets were discarded. The supernatant was centrifuged at 380 x g for 20 min, and the pellet was washed twice with 10 mM citric acid buffer containing 5 mM KCl, 145 mM NaCl, 14 mM glucose, and 1 mM MgCl2, pH 6.4. The obtained platelet pellets were frozen in liquid nitrogen until further use.

Hydrazide Affinity Capture—
Lysis of platelets was performed by adding 15 µl of 1% SDS, 100 mM NaCl, 50 mM Tris, pH 7.8 per milligram of platelet wet weight. 10 glass beads (1.2-mm diameter, VWR International, Nuernberg, Germany) were added for a volume of 1000 µl, and lysis was supported by ultrasonication. After reduction with 25 mM dithiothreitol and alkylation using 60 mM iodoacetamide samples were dialyzed against 100 mM sodium phosphate, pH 7.0 overnight. Sodium periodate was added to a concentration of 4 mg/ml, and incubation was performed at room temperature for 40 min in the dark. After a second dialysis against 100 mM sodium phosphate buffer samples were incubated with 500 µl of hydrazide bead slurry for 7 h at room temperature. Washing the resin with 100 mM urea, 50 mM Tris, pH 7.8 was followed by proteolytic cleavage of trapped glycoproteins with trypsin (0.5 µg/µl) at 37 °C overnight. The supernatant was removed, and the beads were washed thoroughly with 1 M urea in 50 mM Tris, pH 7.8 prior to digestion with PNGaseF (0.02 units/ml) at 37 °C overnight in 100 mM sodium phosphate, pH 7.0.

Concanavalin A Affinity Capture—
For lectin-based enrichment of glycopeptides platelets were lysed in 1.6% n-octylglucoside in buffer A (100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 30 mM Tris, pH 7.4) resulting in 7 mg/ml concentrations of proteins. After 1:1 dilution with buffer A the sample was applied to a self-packed 4.6-mm-inner diameter x 100-mm-long column packed with concanavalin A-derivatized agarose beads equilibrated extensively with buffer A. Samples were eluted with 200 mM {alpha}-methylmannoside in buffer A. Following repeated buffer exchange during repeated ultrafiltration (Centricon; molecular weight cutoff, 5000; Millipore, Bedford, MA) against 20 mM ammonium bicarbonate, pH 7.8 the sample was tryptically digested at 37 °C for 16 h at 80 µg/µl trypsin concentration. Upon deactivation of trypsin by heating to 95 °C for 3 min the sample was diluted 1:1 (v/v) and underwent a second lectin affinity chromatography under similar conditions. The eluate was concentrated, and digestion with PNGaseF (0.02 units/ml) was performed at 37 °C overnight in 100 mM sodium phosphate buffer, pH 7.0.

Reversed-phase Separation of Peptide Mixtures—
Separation of complex peptide mixtures was achieved by using reversed-phase chromatography. For nano-LC-ESI-MS/MS experiments a setup consisting of an autosampler (FamosTM, Dionex, Idstein, Germany) and precolumn concentration (SwitchosTM, Dionex) prior to nano-LC separation (UltimateTM, Dionex) was used. Precolumns (300-µm inner diameter x 1-mm length) and separation columns (75-µm inner diameter x 150-mm length, C18 PepMapTM) were purchased from Dionex. Gradient elution was performed using a linear gradient from 5 to 50% solvent B (84% acetonitrile, 0.1% formic acid) during a period of 2 h. Solvent A was 0.1% formic acid in water. Separation was followed by rinsing the column with 95% B for 5 min prior to equilibration to 5% solvent B before the next separation cycle.

Mass Spectrometric Analysis—
Mass spectrometric analysis was performed on a Qstar® XL system (Applied Biosystems, Framingham, MA) or a Qtrap 4000 linear ion trap system (Applied Biosystems) both equipped with nano-ESI sources. Using distal coated fused silica tips (New Objective, Woburn, MA) spray voltage was set around 2200 V for both systems. A survey scan (m/z 350–2000) was followed by three MS/MS scans fragmenting the three most intensive peptide signals. For the Qstar the acquisition time for both MS and MS/MS scan cycles was 2 s. The Qtrap scan cycle consisted of an enhanced multiple charge survey scan (1 s at 4000 amu/s setting) and a single enhanced resolution scan (0.5 s at 250 amu/s for three precursor ions). Each of the following three enhanced product ion spectra was recorded at 4000 amu/s for 1.5 s each (again two spectra were summed in the process). Exclusion time was set to 16 s for the Qstar XL system and 30 s for the Qtrap 4000.

Data Interpretation—
Experimentally derived mass spectrometric datasets were evaluated by database searches using the MASCOTTM (Version 1.9, Matrix Science, London, UK) search algorithm. Peak lists were generated by the Analyst QS (Qstar® XL) and Analyst 1.4 (Qtrap 4000) software plug-ins (mascot.dll; Matrix Science). Using this script all peaks with intensities below 0.1% of the base peak were omitted in the peak lists, and the data were centroided in the process. Mass deviance was set lower or equal to 0.2 Da to readily identify a 1-Da shift of an Asn to Asp conversion. Searches were conducted using a human subset of the National Center for Biotechnology Information (NCBI) non-redundant database (www.ncbi.nih.gov). Trypsin was used as specific protease with two missed cleavages allowed. Furthermore, searches were repeated without specification of a protease to look for unspecific cleavage products. Only spectra with a matching probability of >95% were selected for manual revision.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Two different techniques were used to reduce the complexity of whole cell lysates: lectin affinity and oxidation/hydrazide trapping of glycopeptides. The carbohydrates were eliminated after the trapping/enrichment, and the thus modified (+1-Da) tryptic peptides were subjected to LC-ESI-MS/MS analysis. 70 individual glycosylation sites on 41 different glycoproteins were assigned from these experiments as presented in Table I. A comparison with the well annotated Swiss-Prot database (www.expasy.org) revealed a high percentage of glycosylation sites not confirmed experimentally before. The database differentiates primarily between known sites with experimental evidence present and potential sites annotated by screening for the NX(S/T) consensus sequence. Furthermore, sites can be annotated as similar by transferring site information between closely related protein family members or be termed as unknown in all other cases. Combining unknown sites, potential sites, and sites determined by similarity studies alone, 69% of the sites have not been determined so far (see Fig. 1). More than half of the identified sites (52%) were previously annotated automatically at every N-glycosylation consensus sequence due to the fact that the protein was known to be N-glycosylated. Furthermore, 10 sites were found on proteins not known to be glycosylated at all or for which the site of glycosylation was not known.


Figure 5
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  		TABLE I N-Glycosylation sites identified within human platelet proteins

Protein names/accession numbers and glycosylation sites are annotated according to Swiss-Prot (SP) database (www.expasy.org). Sites are marked as known, unknown, potential, and by similarity according to Swiss-Prot definition. Sites of unspecific cleavages are marked by asterisks (*). ConA, concanavalin A.

 

Figure 1
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  		FIG. 1. Classification of N-glycosylation sites. The classification in "known," "unknown," "potential" (sites assigned automatically without experimental proof), and "by similarity" is based upon comparison of the experimental dataset with the annotated Swiss-Prot database (www.expasy.org). In total 70 sites could be identified from human platelet proteins.

 
A representative spectrum of a human thrombospondin 1 glycosylation site identified during the course of this study is depicted in Fig. 2. The spectrum clearly exhibits a 1-Da shift of the asparagine to aspartic acid conversion within the sequence (K)VVN*STTGPGEHLR(N) (positions 1065–1077 in the protein sequence; residues in parentheses are adjacent to the found peptide sequence) as well as of the total peptide mass (the asterisk marks the former site of glycosylation at Asn-1067). However, only the fact that a near complete series of y-ions as well as a consensus sequence is present validated the finding of this glycosylation site. In combination with reasonable mass accuracy of the instruments used (<0.2 Da) and defined proteolytic conditions (tryptic digests result in Arg and Lys as C-terminal amino acids; few nonspecific cleavage products are marked with asterisks in Table I) a series of criteria safeguarding the experimental results is present. Furthermore, the sample preparation that ensures an enrichment of glycopeptides within the sample by specific affinity offers a preselection criterion for possible peptide hits. For the most part only the major structural proteins, e.g. actin and filamin, could be observed in the preparations, although they showed strongly reduced abundance in comparison with setups without affinity selection of target peptides, e.g. multidimensional liquid chromatography (data not shown). Together with the more conservative scoring parameters of the MASCOT algorithm the number of false positive identifications could be minimized in this context. Spontaneous deamidation therefore seems rather unlikely for generating the results presented, although it cannot be excluded completely. Most MS/MS spectra giving positive hits were derived from doubly charged precursor ions that resulted predominantly in comprehensive y-ion series. Although a number of 23 triply charged precursors were used for site determination, many more were discarded because the site of glycosylation was often not assigned unambiguously. Therefore, the majority of the 56 identifications could be assigned due to doubly charged precursors.


Figure 2
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  		FIG. 2. Nano-ESI-Q-TOF-MS/MS spectrum of a human thrombospondin 1 peptide. The spectrum identifies a glycosylation site within the peptide sequence VVN*STTGPGEHLR (the asterisk marks the former site of glycosylation). Main features of the spectrum are a 1-Da shift due to the Asn to Asp conversion after enzymatic deglycosylation and a clear series of y-ions. Those have been observed to be typical for this instrument configuration. The high intensity of the y7 ion corresponds to the positioning of a labile proline bond in the peptide sequence.

 
Evaluating the results of the two individual methods of affinity-directed glycosylation site analysis revealed a complementary behavior. Although some overlap is observable, many of the sites were either determined by concanavalin A or hydrazide affinity but not both (see Fig. 3). This distribution reflects certain distinct specificities of the affinity purifications. While concanavalin A accounts for trapping structures containing {alpha}-linked mannose residues, the hydrazide chemistry exhibits a somewhat broader specificity. This is due to the binding of virtually all sugars carrying a vicinal diol group. This group is present in most oligosaccharides derived from biological samples. In total more sites were found with the lectin affinity chromatography, although the ratio of determined sites per protein is in favor of the chemical affinity method.


Figure 3
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  		FIG. 3. Numerical comparison of methods used for N-glycosylation analysis. The majority of sites and proteins were determined by lectin affinity chromatography and to a lesser degree by chemical affinity chromatography (oxidation/hydrazide). The lesser amount of overlapping identifications emphasizes the need for redundant methods to be used. ConA, concanavalin A.

 
The complete list of proteins and sites identified is presented within Table I. To enable easier and more reliable access the identifiers are given as Swiss-Prot entries and not as NCBI non-redundant database accession numbers as derived from the database search. Therefore, all identified protein sequences have been blasted against Swiss-Prot using the NCBI blast utility (12). Proteins contained in the list can be classified into a number of different functions and classes, e.g. receptors, enzymes, and transporter/storage proteins. In addition to the integrins {alpha}2, {alpha}6, and ß3 the membrane-bound receptor G6f was identified in platelets, proving that also membrane-bound and low abundance proteins are detectable using the methods described. As examples for enzymes identified as glycoproteins heparanase and endoplasmin can be cited. Heparanases themselves are essential for catabolism of heparin sulfate proteoglycans. Those enzymes are suspected to assist in degradation of basement membrane heparin sulfate proteoglycans during injury or inflammation (13). Endoplasmin, a molecular chaperone, was also identified during a similar study aimed for the identification of glycosylation sites on human plasma proteins (14). However, only Asn-217 and Asn-445 were presented and annotated in Swiss-Prot, whereas during the course of our study also sites Asn-62 and Asn-107 could be verified. Obviously, because the layout of the experiments as well as the sample material differs to a certain degree, methods addressing the analysis of a complex sample have to be multifaceted.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, a first systematic analysis of asparagine-linked glycosylation sites of human platelet proteins is presented. Although the number of identified proteins is relatively small up to now, it clearly identifies a number of suspected and novel glycosylation sites. Often suspected sites have not been thoroughly investigated but were suggested only by indirect evidence such as gel shift assays (15) and periodic acid-Schiff staining. These methods indicate the presence of glycosylation but not the distinct type or the precise attachment site. In our study, lectin affinity chromatography and hydrazide bead trapping of glycosylated peptides in combination with nano-LC-ESI-MS/MS provided direct proof of glycosylation type and sites while minimizing false positive assignments.

The identified proteins may be classified into a range of functions or localizations. Here, some interesting proteins, properties, and perspectives will be addressed. Despite the fact that many of the proteins displayed in Table I are known for some time, their glycosylation sites have not been characterized in detail. Integrins and other glycoproteins are intensely investigated as key players for cell-cell communication and cell-matrix interactions as well as signal transduction in this context because they are important drug targets. Indeed six glycosylation sites were found on integrins {alpha}2, {alpha}6, and ß3 of which only one was annotated before. Therefore, our data will certainly stimulate (re)investigations of the functional properties of these adhesion molecules. However, the annotation of post-translational modifications in current databases requires improvement in the near future. Although the Swiss-Prot database already features a certain number of glycosylation sites, a still unknown number is missing. Other initiatives, as for example the human protein reference database (16), also annotate validated datasets from the literature to improve the quality of databases. Therefore, the combination of proteomic studies and direct manual input in reference databases may lead to more efficient data storage within sequence databases.

A very interesting example of a new platelet protein found in our present study is G6f. It is shown here as an example for some less known or unknown proteins described in Table I (e.g. CLPTM1, GRGP2438, and EMILIN1). Human G6f is encoded by a gene in the major histocompatibility complex and represents a type I transmembrane protein belonging to the immunoglobulin superfamily. It represents a putative cell surface receptor with an intracellular tyrosine phosphorylation site. Cell culture experiments indicated that G6f interacts, via its Src homology 2 domains, with Grb2 and Grb7 and is involved in downstream signaling of the Ras-mitogen-activated protein kinase pathway (17). Interestingly the G6f receptor has so far not been described in platelets. The possible functional role of G6f in platelets is the topic of ongoing investigations in our laboratory.

As an example of a more common platelet protein, thrombospondin 1, an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions (18), was also identified during our present studies. It binds to fibrinogen, fibronectin, laminin, type V collagen, and integrins {alpha}V/ß1, {alpha}V/ß3, and {alpha}IIb/ß3. It comprises four sites of N-glycosylation of which only Asn-248 has been annotated in the Swiss-Prot database so far. As seen in Fig. 1, an additional site could be assigned to Asn-1067 as well.

Some minor contaminations were also observed, such as plasma proteins, which may adhere to the plasma membrane of platelets. {alpha}1-Antitrypsin, a well characterized protein of the human plasma, was identified alongside two known sites of N-glycosylation (Asn-70 and Asn-271). Clearly, plasma contaminations from this source cannot be excluded completely due to the platelet isolation procedures used which are aimed to maintain platelet function and integrity. However, some proteins are present both in plasma fluid as well as in platelets, e.g. coagulation factor V. This protein is a cofactor that participates with factor Xa to activate prothrombin to thrombin. 75–80% is distributed to plasma fluid, and 20–25% is distributed to {alpha}-granules of platelets (19). Results indicate that its N-linked carbohydrate moieties play a substantial role in activated protein C-catalyzed cleavage and inactivation of coagulation factor V (20). Four sites of N-glycosylation of factor V could be determined unambiguously of which Asn-297 is close to the thrombin cleavage site at Arg-334. Coagulation factor V is closely associated to multimerin 1, a storage protein located in platelet {alpha}-granules (21). This massive glycoprotein (22) has 23 potential glycosylation sites of which nearly 50% could now be validated using a combination of hydrazide and lectin affinity. Following factor V activation the multimerin 1-coagulation factor V complex is dissociated suggesting a role for multimerin 1 in delivering and localizing factor V onto platelets prior to prothrombinase assembly (23).

Regarding the specificity of the capturing methods used, only minor conclusions can be drawn on the carbohydrate moiety of the identified glycopeptides. Being one of the few glycoproteins studied extensively over the years, {alpha}1-antitrypsin was identified during the course of the experiments. The same protein but a different glycosylation site (Asn-271 in the present study and Asn-70 in the study of Zhang et al. (14)) was identified in a previous hydrazide affinity strategy (14). The glycan extensions provided by GlycoSuiteDB (5) comprise a range of complex glycans (24). Those structures can obviously be trapped by the hydrazide chemistry as shown before and also by concanavalin A interactions as demonstrated by Baenziger and Fiete (9). Furthermore, the complexity of carbohydrate diversity within a single cell has to be taken into account. It is possible that differentially processed stages of glycosylated precursors are captured using the two methods. The distribution of sites (Fig. 3) elucidated by the two methods shows a moderate overlap but also high numbers of sites found only by one method or the other. This result may be the outcome of different specificities of the capturing methods but could also be due to the individual sample treatments during the two analysis pathways.

An interesting application for the use of glycosylation site information is given by combination with structural data already existing in form of x-ray-elucidated protein structures. Therefore, a web-based tool, GlyProt (www.glycosciences.de), is already available. It uses protein database (25) entries (from the Protein Data Bank) and attaches N-glycans to user-defined positions within the sequence. The program offers the choice between different conformations of the chosen N-glycan to be attached to the site. Also the use of custom designed glycans is supported. In silico glycosylation is sometimes the only way to gain insight into structural details. Many glycosylated proteins do not crystallize well, and sugars are therefore removed prior to x-ray analysis. The algorithm is based on the use of experimental datasets concerning the linkage between amino acids and sugar moiety (26–28). The structure of the glycan itself is simulated by parts of the Sweet-II program (29, 30). Basically, GlyProt generates possible combinations between the crystallographic data of proteins and the structural features of carbohydrate additions to the polypeptide backbone. In Fig. 4, A and B, the in silico glycosylated structure of the epidermal growth factor P-selectin (Protein Data Bank entry 1G1Q (31)) is displayed together with the unglycosylated variant (Fig. 4C). The epidermal growth factor domain of P-selectin has been connected to several cell adhesion models (32, 33) in the past. Glycosylation at Asn-54 completely changes the interaction surface of the domain structure. The attached oligosaccharide is a high mannose glycan in this case, although for a cell surface glycoprotein this structure may be modified (34). Surface charge differences due to N-acetylneuraminic acid attachment have to be taken into account as well when modeling interaction surfaces or designing experimental setups under those circumstances. It is therefore evident that the elucidation of glycosylation site information yields substantial benefit for ongoing research in this field by adding critical information, e.g. for the design of interaction studies. Moreover, the techniques are mandatory when applied to a cell type devoid of possibilities for genetic engineering. Because platelets possess no nuclei, thus no genomic DNA, the conventional methods for glycosylation analysis, e.g. site-specific mutations of consensus sequences, are not accessible at all. In this case direct analysis of protein modifications is necessary as demonstrated in this report. Tripeptidyl-peptidase I is a valid example for this problem. Its sites of glycosylation have been reviewed in the model system of Chinese hamster ovary and human embryonic kidney 293 cells (35) but not in platelets. Therefore, results gained in the one system may not be readily transferable to the other.


Figure 4
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  		FIG. 4. In silico glycosylation of the epidermal growth factor (EGF) domain of P-selectin. The epidermal growth factor domain has been modified with a high mannose N-glycan at position Asn-54 (which equals Asn-13 in the Protein Data Bank entry 1G1Q). A and B depict ribbon and solid surface images of the domain, respectively, and C shows the epidermal growth factor domain without the glycan.

 
A major drawback of the analysis methods is the substantial loss of information due to the removal of the glycan moiety. Thus, few assumptions can be made on the nature of the oligosaccharide structure. Because PNGaseF cleaves nearly all N-glycans it is not possible to add additional specificity during this process and therefore gain additional information on the detected peptides. However, this problem can be addressed by broadening the range of resins and endoglycosidases used. Lectins with enhanced specificity for e.g. hybrid or complex structures or specific antennae conformations of glycans can be used. Additionally, a combined endoglycosidase digest using e.g. endoglycosidases F2 and H can be performed. While endoglycosidase F2 cleaves biantennary complex and high mannose structures, endoglycosidase H is active against hybrid and oligomannose glycans in contrast. Therefore, the nature of the glycan moiety can be elucidated by comparison of two separate experiments. In conclusion, glycoproteomic studies of platelet proteins are expected to stimulate future (re)investigations of established and novel human platelet proteins that will certainly be helpful to explain some of the heterogeneity of platelet function in health and disease.


  FOOTNOTES
 
Received, October 3, 2005, and in revised form, October 26, 2005.

Published, MCP Papers in Press, October 31, 2005, DOI 10.1074/mcp.M500324-MCP200

1 The abbreviation used is: PNGaseF, peptide:N-glycosidase F. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 49-931-201-48730; Fax: 49-931-201-48123; E-mail: Albert.Sickmann{at}virchow.uni-wuerzburg.de


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maguire, P. B., and Fitzgerald, D. J. (2003 ) Platelet proteomics. J. Thromb. Haemostasis1, 1593 –1601[CrossRef]

  2. Sickmann, A., Reinders, J., Wagner, Y., Joppich, C., Zahedi, R., Meyer, H. E., Schonfisch, B., Perschil, I., Chacinska, A., Guiard, B., Rehling, P., Pfanner, N., and Meisinger, C. (2003 ) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U. S. A.100, 13207 –13212[Abstract/Free Full Text]

  3. Hofsteenge, J., Huwiler, K. G., Macek, B., Hess, D., Lawler, J., Mosher, D. F., and Peter-Katalinic, J. (2001 ) C-Mannosylation and O-fucosylation of the thrombospondin type 1 module. J. Biol. Chem.276, 6485 –6498[Abstract/Free Full Text]

  4. Van den Steen, P., Rudd, P. M., Dwek, R. A., and Opdenakker, G. (1998 ) Concepts and principles of O-linked glycosylation. Crit. Rev. Biochem. Mol. Biol.33, 151 –208[CrossRef][Medline]

  5. Cooper, C. A., Harrison, M. J., Wilkins, M. R., and Packer, N. H. (2001 ) GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources. Nucleic Acids Res.29, 332 –335[Abstract/Free Full Text]

  6. Gu, J., and Taniguchi, N. (2004 ) Regulation of integrin functions by N-glycans. Glycoconj. J.21, 9 –15[CrossRef][Medline]

  7. Tarentino, A. L., Gomez, C. M., and Plummer, T. H., Jr. (1985 ) Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry24, 4665 –4671[CrossRef][Medline]

  8. Tretter, V., Altmann, F., and Marz, L. (1991 ) Peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached {alpha}1->3 to the asparagine-linked N-acetylglucosamine residue. Eur. J. Biochem.199, 647 –652[Medline]

  9. Baenziger, J. U., and Fiete, D. (1979 ) Structural determinants of concanavalin A specificity for oligosaccharides. J. Biol. Chem.254, 2400 –2407[Free Full Text]

  10. Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987 ) Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides. J. Biol. Chem.262, 1294 –1299[Abstract/Free Full Text]

  11. Tandon, N. N., Lipsky, R. H., Burgess, W. H., and Jamieson, G. A. (1989 ) Isolation and characterization of platelet glycoprotein IV (CD36). J. Biol. Chem.264, 7570 –7575[Abstract/Free Full Text]

  12. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997 ) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.25, 3389 –3402[Abstract/Free Full Text]

  13. Bame, K. J. (2001 ) Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans. Glycobiology11, 91R –98R[Abstract/Free Full Text]

  14. Zhang, H., Li, X. J., Martin, D. B., and Aebersold, R. (2003 ) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol.21, 660 –666[CrossRef][Medline]

  15. Patel, O. V., Takahashi, T., Imai, K., and Hashizume, K. (2004 ) Characterization of native and recombinant bovine pregnancy-associated glycoproteins. Res. Vet. Sci.77, 203 –210[Medline]

  16. Peri, S., Navarro, J. D., Amanchy, R., Kristiansen, T. Z., Jonnalagadda, C. K., Surendranath, V., Niranjan, V., Muthusamy, B., Gandhi, T. K., Gronborg, M., Ibarrola, N., Deshpande, N., Shanker, K., Shivashankar, H. N., Rashmi, B. P., Ramya, M. A., Zhao, Z., Chandrika, K. N., Padma, N., Harsha, H. C., Yatish, A. J., Kavitha, M. P., Menezes, M., Choudhury, D. R., Suresh, S., Ghosh, N., Saravana, R., Chandran, S., Krishna, S., Joy, M., Anand, S. K., Madavan, V., Joseph, A., Wong, G. W., Schiemann, W. P., Constantinescu, S. N., Huang, L., Khosravi-Far, R., Steen, H., Tewari, M., Ghaffari, S., Blobe, G. C., Dang, C. V., Garcia, J. G., Pevsner, J., Jensen, O. N., Roepstorff, P., Deshpande, K. S., Chinnaiyan, A. M., Hamosh, A., Chakravarti, A., and Pandey, A. (2003 ) Development of human protein reference database as an initial platform for approaching systems biology in humans. Genome Res.13, 2363 –2371[Abstract/Free Full Text]

  17. De Vet, E. C., Aguado, B., and Campbell, R. D. (2003 ) Adaptor signalling proteins Grb2 and Grb7 are recruited by human G6f, a novel member of the immunoglobulin superfamily encoded in the MHC. Biochem. J.375, 207 –213[CrossRef][Medline]

  18. Adams, J. C. (1997 ) Thrombospondin-1. Int. J. Biochem. Cell Biol.29, 861 –865[CrossRef][Medline]

  19. Tracy, P. B., Eide, L. L., Bowie, E. J., and Mann, K. G. (1982 ) Radioimmunoassay of factor V in human plasma and platelets. Blood60, 59 –63[Abstract/Free Full Text]

  20. Silveira, J. R., Kalafatis, M., and Tracy, P. B. (2002 ) Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C. Biochemistry41, 1672 –1680[Medline]

  21. Hayward, C. P., Bainton, D. F., Smith, J. W., Horsewood, P., Stead, R. H., Podor, T. J., Warkentin, T. E., and Kelton, J. G. (1993 ) Multimerin is found in the {alpha}-granules of resting platelets and is synthesized by a megakaryocytic cell line. J. Clin. Investig.91, 2630 –2639

  22. Hayward, C. P., Warkentin, T. E., Horsewood, P., and Kelton, J. G. (1991 ) Multimerin: a series of large disulfide-linked multimeric proteins within platelets. Blood77, 2556 –2560[Abstract/Free Full Text]

  23. Jeimy, S. B., Woram, R. A., Fuller, N., Quinn-Allen, M. A., Nicolaes, G. A., Dahlback, B., Kane, W. H., and Hayward, C. P. (2004 ) Identification of the MMRN1 binding region within the C2 domain of human factor V. J. Biol. Chem.279, 51466 –51471[Abstract/Free Full Text]

  24. Mega, T., Lujan, E., and Yoshida, A. (1980 ) Studies on the oligosaccharide chains of human {alpha}1-protease inhibitor. II. Structure of oligosaccharides. J. Biol. Chem.255, 4057 –4061[Abstract/Free Full Text]

  25. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000 ) The Protein Data Bank. Nucleic Acids Res.28, 235 –242[Abstract/Free Full Text]

  26. Imberty, A., and Perez, S. (1995 ) Stereochemistry of the N-glycosylation sites in glycoproteins. Protein Eng.8, 699 –709[Abstract/Free Full Text]

  27. Petrescu, A. J., Petrescu, S. M., Dwek, R. A., and Wormald, M. R. (1999 ) A statistical analysis of N- and O-glycan linkage conformations from crystallographic data. Glycobiology9, 343 –352[Abstract/Free Full Text]

  28. Petrescu, A. J., Milac, A. L., Petrescu, S. M., Dwek, R. A., and Wormald, M. R. (2004 ) Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology14, 103 –114[Abstract/Free Full Text]

  29. Bohne, A., Lang, E., and von der Lieth, C. W. (1998 ) W3-Sweet: carbohydrate modeling by internet. J. Mol. Model.4, 33 –43

  30. Bohne, A., Lang, E., and von der Lieth, C. W. (1999 ) SWEET—WWW-based rapid 3D construction of oligo- and polysaccharides. Bioinformatics15, 767 –768[Abstract/Free Full Text]

  31. Somers, W. S., Tang, J., Shaw, G. D., and Camphausen, R. T. (2000 ) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLeX and PSGL-1. Cell103, 467 –479[CrossRef][Medline]

  32. Kansas, G. S., Saunders, K. B., Ley, K., Zakrzewicz, A., Gibson, R. M., Furie, B. C., Furie, B., and Tedder, T. F. (1994 ) A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion. J. Cell Biol.124, 609 –618[Abstract/Free Full Text]

  33. Murphy, J. F., and McGregor, J. L. (1994 ) Two sites on P-selectin (the lectin and epidermal growth factor-like domains) are involved in the adhesion of monocytes to thrombin-activated endothelial cells. Biochem. J.303, 619 –624

  34. Mandic, R., Opper, C., Krappe, J., and Wesemann, W. (2002 ) Platelet sialic acid as a potential pathogenic factor in coronary heart disease. Thromb. Res.106, 137 –141[Medline]

  35. Wujek, P., Kida, E., Walus, M., Wisniewski, K. E., and Golabek, A. A. (2004 ) N-Glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I. J. Biol. Chem.279, 12827 –12839[Abstract/Free Full Text]


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