Ten-Yang Yen

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.

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.

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

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 (C5-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.

Human α1,3/4 Fucosyltransferases CHARACTERIZATION OF HIGHLY CONSERVED CYSTEINE RESIDUES ANDN-LINKED GLYCOSYLATION SITES

Human α1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose (Holmes, E. H., Xu, Z., Sherwood, A. L., and Macher, B. A. (1995)J. Biol. Chem. 270, 8145–8151). The participation of the highly conserved Cys residues in disulfide bonds and their functional significance were characterized by mass spectrometry (MS) analyses and site-directed mutagenesis, respectively. Among the human FucTs is a subset of enzymes (FucT III, V, and VI) having highly homologous sequences, especially in the catalytic domain, and Cys residues in FucT III and V were characterized. The amino acid sequence of FucT III was characterized. Peptides containing the four conserved Cys residues were detected after reduction and alkylation, and found to be involved in disulfide bonds. The disulfide bond pattern was characterized by multiple stage MS analysis and the use of Glu-C protease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys residues (Cys81 to Cys338 and Cys91 to Cys341) at the N and C termini of the catalytic domain, bringing these ends close together in space. Mutagenesis of highly conserved Cys residues to Ser in FucT V resulted in proteins lacking enzymatic activity. Three of the four mutants have molecular weights similar to wild type enzyme and maintained an ability to bind GDP, whereas the other (Cys104) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs have highly conserved, potentialN-linked sites, and our mass spectrometry analyses demonstrated that both N-linked sites are modified with oligosaccharides.

Identification of the ZPC Oligosaccharide Ligand Involved in Sperm Binding and the Glycan Structures of Xenopus laevis Vitelline Envelope Glycoproteins

Abstract 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 β-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal β-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain β-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.

Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains.

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys429 and Cys476 forming a disulfide-bonded pair while Cys80 and Cys82 are disulfide bonded in combination with Cys412 and Cys529. Partial reduction to produce monomers converted Cys80 and Cys82 to free thiols while the Cys429 to Cys476 disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys80 and Cys82 to Ser produced monomers but not dimers. In summary these results demonstrate that Cys429 and Cys476 form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys80 and Cys82 with Cys412 and Cys529 are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

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.

An efficient algorithmic approach for mass spectrometry-based disulfide connectivity determination using multi-ion analysis.

BackgroundDetermining the disulfide (S-S) bond pattern in a protein is often crucial for understanding its structure and function. In recent research, mass spectrometry (MS) based analysis has been applied to this problem following protein digestion under both partial reduction and non-reduction conditions. However, this paradigm still awaits solutions to certain algorithmic problems fundamental amongst which is the efficient matching of an exponentially growing set of putative S-S bonded structural alternatives to the large amounts of experimental spectrometric data. Current methods circumvent this challenge primarily through simplifications, such as by assuming only the occurrence of certain ion-types (b-ions and y-ions) that predominate in the more popular dissociation methods, such as collision-induced dissociation (CID). Unfortunately, this can adversely impact the quality of results.MethodWe present an algorithmic approach to this problem that can, with high computational efficiency, analyze multiple ions types (a, b, bo, b*, c, x, y, yo, y*, and z) and deal with complex bonding topologies, such as inter/intra bonding involving more than two peptides. The proposed approach combines an approximation algorithm-based search formulation with data driven parameter estimation. This formulation considers only those regions of the search space where the correct solution resides with a high likelihood. Putative disulfide bonds thus obtained are finally combined in a globally consistent pattern to yield the overall disulfide bonding topology of the molecule. Additionally, each bond is associated with a confidence score, which aids in interpretation and assimilation of the results.ResultsThe method was tested on nine different eukaryotic Glycosyltransferases possessing disulfide bonding topologies of varying complexity. Its performance was found to be characterized by high efficiency (in terms of time and the fraction of search space considered), sensitivity, specificity, and accuracy. The method was also compared with other techniques at the state-of-the-art. It was found to perform as well or better than the competing techniques. An implementation is available at: http://tintin.sfsu.edu/~whemurad/disulfidebond.ConclusionsThis research addresses some of the significant challenges in MS-based disulfide bond determination. To the best of our knowledge, this is the first algorithmic work that can consider multiple ion types in this problem setting while simultaneously ensuring polynomial time complexity and high accuracy of results.

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 proteins 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.