Detection of cadherin-17 in human colon cancer LIM1215 cell secretome and tumour xenograft-derived interstitial fluid and plasma.

Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the 'gold standard' for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome.

PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis.

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.

A fluorescent microsphere-based method for assay of multiple analytes in plasma.

Measurement of multiple analytes can provide increased sensitivity and specificity for the detection and management of disease. The enzyme-linked immunosorbent assay (ELISA) is currently the "gold standard" for protein quantification; however, individual assays for each analyte must be performed, placing demand on sample volume. On the contrary, multiplex assays using microsphere-based technologies allow for multiple analytes to be simultaneously assayed within a single sample. Here, we present a protocol for the preparation and development of a multiple-analyte assay in human plasma using the BioPlex 200 platform (Bio-Rad), which incorporates xMAP technology (Luminex).

Oligomerization of the macrophage mannose receptor enhances gp120-mediated binding of HIV-1.

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.

Proteomics-driven cancer biomarker discovery: looking to the future.

Availability of a suite of biomarkers for early detection, stratification into distinct subtypes, and monitoring progression or response to therapy promises significant improvements in clinical outcomes for cancer patients. However, despite the recent progress in proteomics technologies based on mass spectrometry (MS), discovery of novel clinical assessment tools has been slow. This is, partly due to the inherent difficulties in working with blood as the biospecimen for candidate discovery. A better understanding of the limitations of blood for comparative protein profiling and a better appreciation of the advantages of cancer tissue or cancer cell secretomes have the potential to greatly enhance the progress.

Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides.

A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.

New insights into viral structure and virus-cell interactions through proteomics.

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus-cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV-cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.

Proteomic analysis of DC-SIGN on dendritic cells detects tetramers required for ligand binding but no association with CD4.

DC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV.

Analysis of proteins copurifying with the CD4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods.

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies.

Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry.

Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.

Mass spectrometry analysis of CD4-associating proteins using affinity chromatography and affinity tag-mediated purification of tryptic peptides.

The study of protein interactions using mass spectrometry (MS) for identification of the components of purified protein complexes is leading to the description of increasingly valuable data on protein function. Commonly proteins in a given complex are identified via MS analysis of in-gel digests of gel electrophoretically separated proteins. In this study, we have evaluated the use of an approach employing the digest of the whole protein complex to identify directly the proteins present in a purification of the CD4 receptor complex. We used a cysteinyl affinity capture method to reduce the complexity of the peptide mixture that was obtained from the tryptic digest of the whole protein complex to the rather limited mixture of only cysteine-containing peptides. Here we report the use of this approach with MS for identification of the CD4 receptor complex components CD4 and p56lck, along with several other proteins present in the detergent-solubilized fractions from the purification. We have been able to identify these proteins using peptide sequence data obtained from cysteine-containing peptides. With appropriate control experiments, we have demonstrated the specific nature of the CD4-p56lck interaction. In contrast, the other proteins identified are shown to arise from nonspecific interactions during the affinity chromatography purification suggesting a possible loss of specific interactions during the chromatography procedure. We found that the complexity of the mixture was reduced such that only 10% of the peptides derived from tryptic digest of the identified proteins were detected. This represents only one-third of the cysteine-containing peptides, however, suggesting that this approach does not enable detection of all individual proteins.