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.

Naphthablins B and C, Meroterpenoids Identified from the Marine Sediment-Derived Streptomyces sp. CP26-58 Using HeLa Cell-Based Cytological Profiling.

HeLa cell-based cytological profiling (CP) was applied to an extract library of marine sediment-derived actinomycetes to discover new cytotoxic secondary metabolites. Among the hit strains, Streptomyces sp. CP26-58 was selected for further investigation to identify its cytotoxic metabolites. CP revealed that the known ionophore tetronasin (1) was responsible for the cytotoxic effect found in the extract. Furthermore, three naphthoquinone meroterpenoids, naphthablin A (2) and two new derivatives designated as naphthablins B (3) and C (4), were isolated from other cytotoxic fractions. The structures of the new compounds were elucidated based on analysis of their HRESIMS and comprehensive NMR data. The absolute configurations of the new compounds were deduced by simulating ECD spectra and calculating potential energies for the model compounds using density function theory (DFT) calculations. Compound 1 showed a significant cytotoxic effect against HeLa cells with an IC50 value of 0.23 µM, and CP successfully clustered 1 with calcium ionophores.

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.

Proteomic analysis of the extracellular matrix produced by mesenchymal stromal cells: implications for cell therapy mechanism.

Mesenchymal stromal cells (MSCs) transiently transfected with notch1 intracellular domain (NICD) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from NICD-transfected MSCs has been previously shown to support in vitro neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which NICD-transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in NICD-transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and NICD-transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from NICD-transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing in vitro cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy.

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.

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

BACKGROUND: Determining 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. METHOD: We 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. RESULTS: The 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. CONCLUSIONS: This 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.

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.

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.

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.

An algorithmic approach to automated high-throughput identification of disulfide connectivity in proteins using tandem mass spectrometry.

Knowledge of the pattern of disulfide linkages in a protein leads to a better understanding of its tertiary structure and biological function. At the state-of-the-art, liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) can produce spectra of the peptides in a protein that are putatively joined by a disulfide bond. In this setting, efficient algorithms are required for matching the theoretical mass spaces of all possible bonded peptide fragments to the experimentally derived spectra to determine the number and location of the disulfide bonds. The algorithmic solution must also account for issues associated with interpreting experimental data from mass spectrometry, such as noise, isotopic variation, neutral loss, and charge state uncertainty. In this paper, we propose a algorithmic approach to high-throughput disulfide bond identification using data from mass spectrometry, that addresses all the aforementioned issues in a unified framework. The complexity of the proposed solution is of the order of the input spectra. The efficacy and efficiency of the method was validated using experimental data derived from proteins with with diverse disulfide linkage patterns.

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.

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.

Identification of specific protein carbonylation sites in model oxidations of human serum albumin.

Human serum albumin (HSA) was subjected to oxidative stress and the locations of the resulting protein carbonyls were determined using mass spectrometry in conjunction with a hydrazide labeling scheme. To model oxidative stress, HSA samples were subjected to metal-catalyzed oxidation (MCO) conditions or treated with hypochlorous acid (HOCl). Oxidation led to the conversion of lysine residues to 2-aminoadipic semi-aldehyde residues, which were subsequently labeled with biotin hydrazide. Analysis of the tryptic peptides from the samples indicates that the oxidations are highly selective. Under MCO conditions, only two of the 59 lysine residues appeared to be modified (Lys-97 and Lys-186). With HOCl, five different lysine modification sites were identified (Lys-130, Lys-257, Lys-438, Lys-499, and Lys-598). These results strongly suggest that the preferred site of modification is dependent on the nature of the oxidant and that the process relies on specific structural motifs in the protein to direct the oxidation. The high selectivity seen here provides insights into the factors that in vivo drive the selective carbonylation of specific proteins in systems under oxidative stress.

Structural features of the lysosomal hydrolase mannose 6-phosphate uncovering enzyme.

The uncovering enzyme (UCE) removes N-acetylglucosamine from lysosomal enzymes to uncover the mannose 6-phosphate (Man-6-P) determinant necessary for targeting these enzymes to lysosomes. Failure to create the Man-6-P determinant is one cause of lysosomal storage diseases. Despite its medical importance, little structural information about UCE is available. In this report we have developed a model for the membrane proximal portion of the lumenal domain of UCE based on the structure of the EFG-3 and -4 domains of the extracellular segment of the beta chain of integrin alpha Vbeta 3. In this model the EGF-like domains of UCE (residues 285-345) are predicted to form a rod-shaped stalk region, similar to the stem region in Golgi glycosyltransferases. This stalk causes the proposed catalytic domain (residues 1-277) to be extended away from the Golgi membrane. A portion of the proposed catalytic domain (residues 85-256) resides in Cluster of Orthologous Group (COG) 4632 with four bacterial proteins but is not homologous to any known eukaryotic proteins. Thus, UCE may have evolved from the fusion of a unique catalytic domain with a common EGF-like stalk domain. We have determined by mass spectrometry that the four disulfide bonds of the proposed catalytic domain are located between Cys(2)-Cys(172), Cys(66)-Cys(99), Cys(83)-Cys(274), and Cys(258)-Cys(265). Finally, we determined that four of the six potential N-linked glycosylation sites are glycosylated (Asn 159, Asn 165, Asn 247, and Asn 317) in COS cells.

Characterization of the disulfide bonds and free cysteine residues of the Chlamydia trachomatis mouse pneumonitis major outer membrane protein.

Members of the genus Chlamydia lack a peptidoglycan layer. As a substitute for peptidoglycan, it has been proposed that several cysteine rich proteins, including the major outer membrane protein (MOMP), form disulfide bonds to provide rigidity to the cell wall. Alignment of the amino acids sequences of the MOMP from various serovars of Chlamydia showed that they have from 7 to 10 cysteine residues and seven of them are highly conserved. Which of these are free cysteine residues and which are involved in disulfide bonds is unknown. The complexity of the outer membrane of Chlamydia precludes at this point the characterization of the structure of the cysteines directly in the bacteria. Therefore, mass spectrometric analysis of a purified and refolded MOMP was used in this study. Characterization of the structure of this preparation of the MOMP is critical because it has been shown, in an animal model, to be a very effective vaccine against respiratory and genital infections. Here, we demonstrated that in this MOMP preparation four cysteines are involved in disulfide bonds, with intramolecular pairs formed between Cys(48) and Cys(55) and between Cys(201) and Cys(203). A stepwise alkylation, reduction, alkylation process using two different alkylating reagents was required to establish the Cys(48)-Cys(55) disulfide pair. The other residues in MOMP, Cys(51), Cys(136), Cys(226), and Cys(351), are free cysteines and could potentially form disulfide-linked complexes with other MOMP or other membrane proteins.