Over-Expression of RNA Processing, Heat Shock, and DNA Repair Proteins in Breast Tumor Compared to Normal Tissue.

This study identifies the main changes in protein expression in human breast tumors compared to normal breast tissue. Malignant tumors (32) and normal breast tissue samples (23), from formaldehyde-fixed, paraffin-embedded specimens are subjected to discovery proteomics using liquid chromatography/tandem mass spectrometry, with spectral counts for quantitation. The dataset contains 1406 proteins. Differential expression is measured using a method that takes advantage of estimates of the percentage of tumor on a slide. This analysis shows that the major classes of proteins over-expressed by tumors are RNA-binding, heat shock and DNA repair proteins. RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins (HNRNPs), SR splice factors (SRSF) and elongation factors form the largest group. Comparison with results from another study demonstrates that the RNA-binding proteins are associated specifically with malignant transformation, rather than with cell proliferation. HNRNP and SRSF proteins help define splice sites in normal cells. Their over-expression may dysregulate splicing, which in turn has the potential to promote malignant transformation.

Glycoproteins in Claudin-Low Breast Cancer Cell Lines Have a Unique Expression Profile.

Claudin proteins are components of epithelial tight junctions; a subtype of breast cancer has been defined by the reduced expression of mRNA for claudins and other genes. Here, we characterize the expression of glycoproteins in breast cell lines for the claudin-low subtype using liquid chromatography/tandem mass spectrometry. Unsupervised clustering techniques reveal a group of claudin-low cell lines that is distinct from nonmalignant, basal, and luminal lines. The claudin-low cell lines express F11R, EPCAM, and other proteins at very low levels, whereas CD44 is expressed at a high level. Comparison of mRNA expression to glycoprotein expression shows modest correlation; the best agreement occurs when the mRNA expression level is lowest and little or no protein is detected. These findings from cell lines are compared to those for tumor samples by the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CPTAC samples contain a group low in CLDN3. The samples low in CLDN3 proteins share many differentially expressed glycoproteins with the claudin-low cell lines. In contrast to the situation for cell lines or patient samples classified as claudin-low by RNA expression, however, most of the tumor samples low in CLDN3 protein express the estrogen receptor or HER2. These tumor samples express CD44 protein at low rather than high levels. There is no correlation between CLDN3 gene expression and protein expression in these CPTAC samples; hence, the claudin-low subtype defined by gene expression is not the same group of tumors as that defined by low expression of CLDN3 protein.

Mining the Breast Cancer Proteome for Predictors of Drug Sensitivity.

Approximately 20 drugs have been approved by the FDA for breast cancer treatment, yet predictive biomarkers are known for only a few of these. The identification of additional biomarkers would be useful both for drugs currently approved for breast cancer treatment and for new drug development. Using glycoprotein expression data collected via mass spectrometry, in conjunction with statistical models constructed by elastic net or lasso regression, we modeled quantitatively the responses of breast cancer cell lines to ~90 drugs. Lasso and elastic net regression identified HER2 as a predictor protein for lapatinib, afatinib, gefitinib and erlotinib, which target HER2 or the EGF receptor, thus providing an internal control for the approach. Two additional protein datasets and two RNA datasets were also tested as sources of predictor proteins for modeling drug sensitivity. Protein expression measured by mass spectrometry gave models with higher coefficients of determination than did reverse phase protein array (RPPA) predictor data. Further, cross validation of the elastic net models shows that, for many drugs, the prediction error is lower when the predictor data is from proteins, rather than mRNA expression measured on microarrays. Drugs that could be modeled effectively include PI3K inhibitors, Akt inhibitors, paclitaxel and docetaxel, rapamycin, everolimus and temsirolimus, gemcitabine and vinorelbine. Strikingly, this modeling approach with protein predictors often succeeds for drugs that are targeted agents, even when the nominal target is not in the dataset.

Using a cell line breast cancer progression system to identify biomarker candidates.

Secreted and plasma membrane glycoproteins are considered excellent candidates for disease biomarkers. Herein we describe the identification of secreted and plasma membrane glycoproteins that are differentially expressed among a family of three breast cancer cell lines that models the progression of breast cancer. Using two-dimensional liquid chromatography-tandem mass spectrometry we identified more than 40 glycoproteins that were differentially expressed in either the premalignant (MCF10AT) or the fully malignant (MCF10CA1a) cell lines of this model system. Comparative analysis revealed that the differentially expressed breast cancer progression-associated glycoproteins were among the most highly expressed in the malignant (MCF10CA1a) breast cancer cell line; a subset of these was detected only in the malignant line; and others were detected in the malignant line at levels 25 to 50 times greater than in the benign (MCF10A) line. Using the results from this model cell system as a guide, we then carried out glycoproteomic analyses of normal and cancerous breast tissue lysates. Eleven of the glycoproteins differentially expressed in the breast cell lines were identified in the tissue lysates. Among these glycoproteins, collagen alpha-1 (XII) chain was expressed at dramatically higher (~10-fold) levels in breast cancer than in normal tissue. BIOLOGICAL SIGNIFICANCE: Identifying glycoproteins differentially expressed during cancer progression results in information on the biological processes and key pathways associated with cancer. In addition, new hypotheses and potential biomarkers result from these glycoproteomic studies. Our glycoproteomic analysis of this model of breast cancer provides a roadmap for future experimental interventions to further tease apart critical components of tumor progression.

Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines.

Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.

Overcoming challenges and opening new opportunities in glycoproteomics.

Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they are expressed in relatively low abundance in cells. In response, various enrichment methods have been developed to improve the detection of glycoproteins. One such method involves their capture via oxidation of their glycan chains and covalent attachment with hydrazide resins which, when catalyzed by PNGase F, release N-linked glycans and convert the glycosite Asn to Asp; this conversion is identifiable with LC/ESI-MS/MS as a corresponding increase of 0.984 Da in molecular weight. The present study builds on this body of work, providing evidence of three additional strategies that improve glycoprotein identification: (1) use of a high resolution mass spectrometer-the Q Exactive MS-which delivers 2-3 times more glycoprotein identifications than a low resolution MS; (2) optimization of instrument settings and database search parameters to reduce misidentification of N-linked glycopeptides to ~1 percent; and (3) labeling glycopeptides with (18)O during PNGase F treatment to locate N-linked glycosites within peptides containing multiple N-linked sequons.

Glycoprotein profiles of human breast cells demonstrate a clear clustering of normal/benign versus malignant cell lines and basal versus luminal cell lines.

Gene expression profiling has defined molecular subtypes of breast cancer including those identified as luminal and basal. To determine if glycoproteins distinguish various subtypes of breast cancer, we obtained glycoprotein profiles from 14 breast cell lines. Unsupervised hierarchical cluster analysis demonstrated that the glycoprotein profiles obtained can serve as molecular signatures to classify subtypes of breast cancer, as well as to distinguish normal and benign breast cells from breast cancer cells. Statistical analyses were used to identify glycoproteins that are overexpressed in normal versus cancer breast cells, and those that are overexpressed in luminal versus basal breast cancer. Among the glycoproteins distinguishing normal breast cells from cancer cells are several proteins known to be involved in cell adhesion, including proteins previously identified as being altered in breast cancer. Basal breast cancer cell lines overexpressed a number of CD antigens, including several integrin subunits, relative to luminal breast cancer cell lines, whereas luminal breast cancer cells overexpressed carbonic anhydrase 12, clusterin, and cell adhesion molecule 1. The differential expression of glycoproteins in these breast cancer cell lines readily allows the classification of the lines into normal, benign, malignant, basal, and luminal groups.

Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a set of glycoprotein biomarker candidates for thyroid cancer is proposed.

Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome.

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.

Decreased ceramide transport protein (CERT) function alters sphingomyelin production following UVB irradiation.

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

The Galalpha1,3Galbeta1,4GlcNAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance.

In 1985, we reported that a naturally occurring human antibody (anti-Gal), produced as the most abundant antibody (1% of immunoglobulins) throughout the life of all individuals, recognizes a carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc-R (the alpha-gal epitope). Since that time, an extensive literature has developed on discoveries related to the alpha-gal epitope and the anti-Gal antibody, including the barrier they form in xenotransplantation and their reciprocity in mammalian evolution. This review covers these topics and new avenues of clinical importance related to this unique antigen/antibody system (alpha-gal epitope/anti-Gal) in improving the efficacy of viral vaccines and in immunotherapy against cancer.

Proteins at membrane surfaces-a review of approaches.

Membrane proteins are critical for normal cellular differentiation and function, and alterations in these proteins often leads to cell dysfunction and disease. Membrane proteomics aims to identify the membrane protein constituents, their posttranslational modifications, protein-protein interactions, and dynamics. Efforts to identify membrane proteins and elucidate their dynamics have been plagued by the challenges presented by studying water insoluble proteins that are distributed among a range of membranes in a cell and often occur at a relatively low abundance. This brief review presents a summary of the literature related to membrane proteomics with an emphasis on efforts to develop effective protocols for the enrichment of membrane proteins, particularly those located in the plasma membrane.

Site-specific fucosylation of sialylated polylactosamines by alpha1,3/4-fucosyltransferases-V and -VI Is defined by amino acids near the N terminus of the catalytic domain.

Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAcalpha2-3Galbeta1-4Glc-NAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-ceramide leads to two isomeric monofucosyl antigens, VIM2 and sialyl-Le(x). Human alpha1,3/4-fucosyltransferase (FucT)-V catalyzes primarily the synthesis of VIM2, whereas human FucT-VI catalyzes primarily the synthesis of sialyl-Le(x). Thus, these two enzymes have distinct "site-specific fucosylation" properties. Amino acid sequence alignment of these enzymes showed that there are 24 amino acid differences in their catalytic domains. Studies were conducted to determine which of the amino acid differences are responsible for the site-specific fucosylation properties of each enzyme. Domain swapping (replacing a portion of the catalytic domain from one enzyme with an analogous portion from the other enzyme) demonstrated that site-specific fucosylation was defined within a 40-amino acid segment containing 8 amino acid differences between the two enzymes. Site-directed mutagenesis studies demonstrated that the site-specific fucosylation properties of these enzymes could be reversed by substituting 4 amino acids from one sequence with the other. These results were observed in both in vitro enzyme assays and flow cytometric analyses of Chinese hamster ovary cells transfected with plasmids containing the various enzyme constructs. Modeling studies of human FucT using a structure of a bacterial fucosyltransferase as a template demonstrated that the amino acids responsible for site-specific fucosylation map near the GDP-fucose-binding site. Additional enzyme studies demonstrated that FucT-VI has approximately 12-fold higher activity compared with FucT-V and that the Trp(124)/Arg(110) site in these enzymes is responsible primarily for this activity difference.

Site-directed mutagenesis of glutamate 317 of bovine alpha-1,3Galactosyltransferase and its effect on enzyme activity: implications for reaction mechanism.

Bovine alpha1,3galactosyltransferase (alpha1,3GalT) transfers galactose from UDP-alpha-galactose to terminal beta-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar-enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of alpha1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the alpha1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.

Determination of glycosylation sites and disulfide bond structures using LC/ESI-MS/MS analysis.

Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been solved, and very little is known about the structures of membrane proteins. One of the important structural elements of a protein is its disulfide bonds. These covalent bonds place conformational constraints on the overall protein structure, and thus, their identification provides important structural information. A second important posttranslational modification found in proteins is N-linked glycosylation. Although potential sites of N-linked glycosylation can be predicted from a protein's primary sequence based on the presence of N-X-S/T sequences, not all of the predicted sites will be glycosylated. Therefore, N-linked glycosylation sites must be located by structural analysis. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide-bonded Cys residues, as well as the N-linked glycosylation sites in glycoproteins by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the programs Sequest and Mascot. The details of our method are described in this chapter.

Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates.

Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity.

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins.

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.

Eukaryotic glycosyltransferases: cysteines and disulfides.

Significant progress has been made in discovering and cloning a host of eukaryotic glycosyltransferases, demonstrating the intricacy and complexity of protein and lipid glycosylation. The availability of their predicted amino acid sequences extended our insights into the structure/function aspects of this family of proteins. However, our knowledge of their three-dimensional structures and how structure gives rise to substrate binding and specificity is still limited. Glycosyltransferase X-ray crystal structures have begun to provide significant information on a limited number of enzymes. To date, only three eukaryotic glycosyltransferase crystal structures have been solved, and all of them are for enzymes that utilize a UDP-sugar. One of the important structural elements of a protein is its disulfide bonds. These covalent interactions place conformational constraints on the overall protein structure,providing some important structural information. In this letter, we outline our current understanding of the free Cys residues and disulfide bonds in eukaryotic glycosyltransferases and discuss some of the important outcomes of these findings.

Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Identifying the Cys residues involved in disulfide linkages of peptides and proteins that contain complex disulfide bond patterns is a significant analytical challenge. This is especially true when the Cys residues involved in the disulfide bonds are closely spaced in the primary sequence. Peptides and proteins that contain free Cys residues located near disulfide bonds present the additional problem of disulfide shuffling via the thiol-disulfide exchange reaction. In this paper, we report a convenient method to identify complex disulfide patterns in peptides and proteins using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with partial reduction by tris(2-carboxyethyl)phosphine (TCEP). The method was validated using well-characterized peptides and proteins including endothelin, insulin, alpha-conotoxin SI and immunoglobulin G (IgG2a, mouse). Peptide or protein digests were treated with TCEP in the presence of an alkylation reagent, maleimide-biotin (M-biotin) or N-ethylmaleimide (NEM), followed by complete reduction with dithiothreitol and alkylation by iodoacetamide (IAM). Subsequently, peptides that contained alkylated Cys were analyzed by capillary LC/ESI-MS/MS to determine which Cys residues were modified with M-biotin/NEM or IAM. The presence of the alkylating reagent (M-biotin or NEM) during TCEP reduction was found to minimize the occurrence of the thiol-disulfide exchange reaction. A critical feature of the method is the stepwise reduction of the disulfide bonds and the orderly, sequential use of specific alkylating reagents.