Lambert C. Loh

Tumor-associated antigen 90K/Mac-2-binding protein : Possible role in colon cancer

The tumor‐associated antigen 90K (TAA90K)/Mac‐2‐binding protein implicated in cancer progression and metastasis is modified by β1‐6 branched N‐linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K‐His purified from HT‐29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C‐terminal poly‐histidine tag. TAA90K‐His bound to fibronectin, collagen IV, laminins‐1, ‐5, and ‐10 and galectin‐3 (Mac‐2) but poorly to collagen I and galectin‐1. As expected, binding of TAA90K to galectin‐3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K‐His glycoform purified from HT‐29 cells treated with the glycosylation inhibitor 1‐deoxymannojirimycin bound poorly to galectin‐3. Unlike TAA90K isolated from other cell types, TAA90K‐His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell‐type specific glycosylation of TAA90K‐His and/or its putative cellular receptor. However, at low concentrations, TAA90K‐His enhanced galectin‐3‐mediated HT‐29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin‐3. J. Cell. Biochem. 98: 1351–1366, 2006.

Mouse Cytomegalovirus M33 Is Necessary and Sufficient in Virus-Induced Vascular Smooth Muscle Cell Migration

ABSTRACT Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.

Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus

Three glycoproteins on the murine cytomegalovirus (MCMV) virion with apparent molecular weights of 150K (gp 150), 105K (gp 105), and 52K (gp52) were immunoprecipitated by two monoclonal antibodies (MAbs) 8G5.12A and 2E.12A. However, only 8G5.12A was able to neutralize MCMV infectivity in the presence of complement. The accessibility of these three glycoproteins to radiolabeling by surface-iodination reactions suggested that they were exposed on the surface of the virion. Western blot analysis of the three glycoproteins showed that gp150 shared antigenic determinants with gp105 and gp52. Briefly, the MAb 8G5.12A reacted with gp150 and gp105, whereas the MAb 2E8.12A reacted with gp150 and gp52. A third MAb 3H2.12A was also found to be reactive with gp150 and gp105 in Western blots, but was unable to immunoprecipitate these glycoproteins. Data from pluse-chase experiments suggested that all three virion glycoproteins were synthesized from a common 128K precursor, providing a partial explanation of their antigenic relatedness. Furthermore, we have demonstrated the presence of high-molecular-weight complexes formed by disulfide bonding between gp150, gp105, and gp52. Lastly, the MAb 8G5.12A was able to immunoprecipitate 84K and 99-110K glycoproteins from human CMV-infected WI-38 cells, demonstrating that conserved determinants exist between murine and human CMV envelope glycoproteins.

A neutralizing monoclonal antibody recognizes an 87K envelope glycoprotein on the murine cytomegalovirus virion

An 87K glycoprotein (gp87) on the murine cytomegalovirus (MCMV) virion was immunoprecipitated by the neutralizing monoclonal antibody (MAb) 8D1.11A. The 87K glycoprotein is also radiolabeled in a surface iodination reaction, suggesting that it is exposed on the surface of the virion. Using a nondenaturing system of polyacrylamide gel electrophoresis in combination with Western blotting, we have shown that the epitope recognized by the MAb 8D1.11A resides on gp87. The failure of 8D1.11A to react with gp87 in a reduced and denatured form suggests that the epitope is recognized only when disulfide linkages are preserved. Our data also indicated that gp87 is present in the MCMV virion both in a monomeric form and as a component of disulfide-linked complexes. Using a two-dimensional gel electrophoresis system, we have demonstrated the presence of disulfide linkages between gp87 and virion polypeptides with apparent molecular weights of 138K, 46K, and 20K. Finally, the difference in migration rates of gp87 in SDS-polyacrylamide gels under reducing and nonreducing conditions suggests the existence of intramolecular disulfide bonds.

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage.

BACKGROUND The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9. METHODS The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis. RESULTS TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium. CONCLUSION TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells. GENERAL SIGNIFICANCE Proteolytic cleavage of TAA90K may have functional implications in colon cancer.

Monoclonal antibodies specific for human tumor‐associated antigen 90K/Mac‐2 binding protein: Tools to examine protein conformation and function

As part of our effort to identify glycoproteins that contribute to colon cancer progression, we have previously described a family of structurally related glycoproteins expressing β1–6 branched asparagine(Asn)‐linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferté and Loh [1992]; Biochem J; 283:193–201). MAb 1H9 immunoprecipitates three glycoproteins having apparent sizes of 92–100, 66–70, and 25 kDa, the size heterogeneity attributable to cell‐type specific glycosylation differences. We report on the basis of partial protein and cDNA sequence information, that the 100‐kDa glycoprotein detected by MAb 1H9 is identical to the 90‐kDa glycoprotein variably known as tumor‐associated antigen 90K (TAA90K), Mac‐2 binding protein, and cyclophilin C‐associated protein. Using a PCR‐based cloning strategy, the complete cDNA encoding TAA90K was cloned into the eukaryotic expression vector pCDNA‐3 (pCD‐TAA90Kwt) and the protein expressed in COS‐1 cells. A [35S]methionine‐labeled 60‐kDa polypeptide, processed to an endoglycosidase H‐sensitive 74‐kDa glycoprotein in the presence of dog pancreas microsomes, was detected in a coupled transcription/translation in vitro reaction. The in vitro‐translated 60‐kDa polypeptide and N‐glycanase‐treated TAA90K (60‐kDa species) immunoprecipitated from HT29 cells were shown to be structurally identical by limited proteolytic peptide mapping. Using a new panel of 11 TAA90K‐specific monoclonal antibodies, including five specific for human TAA90K and six cross‐reactive with a 90‐kDa species expressed by COS‐1 cells, we have detected conformational differences between recombinant wild‐type TAA90K, in vitro‐synthesized TAA90K, and mutant forms of TAA90K containing point mutations at residues 189, 223, and 259. Furthermore, we have shown that these mutant forms of TAA90K, as well as a truncated form of TAA90K containing amino acid residues 1–383, are defective in secretion. These studies demonstrate the potential usefulness of TAA90K‐specific monoclonal antibodies for examining the structure and function of TAA90K, and highlight the contribution of specific amino acid residues to its normal processing and secretion. J. Cell. Biochem. 77:540–559, 2000.

Characterization of a membrane-associated phosphoprotein of murine cytomegalovirus (pp50) and its immunological cross-reactivity with a human cytomegalovirus protein

We have identified an abundant 50K phosphoprotein (pp50) in MCMV-infected 3T3-L1 cells and shown by immunofluorescence microscopy and surface-iodination experiments that pp50 is localized to the plasma membrane of the infected cell. Furthermore, the kinetics of its synthesis suggests that it belongs to the early-late class of herpesvirus proteins. Using monoclonal antibodies specific for pp50 to screen a lambda ZAP II expression library constructed from poly(A)+ mRNA of MCMV-infected cells, we have isolated a cDNA clone that synthesizes a truncated form of pp50 as a beta-galactosidase fusion protein. This allowed us to localize the partial pp50 transcript to a region between map coordinates 0.228 and 0.260 of the MCMV genome (Smith strain, Vancouver). Finally, we demonstrated that the MAb 5H10.21A recognizes an antigenic determinant that is conserved between pp50 and a 50K human cytomegalovirus (HCMV) nonstructural protein.

Transcriptional analysis of human cytomegalovirus and rat cytomegalovirus homologues of the M73/M73.5 spliced gene family

Previous analysis of the M73-to-m74/M75 intergenic region of murine cytomegalovirus (MCMV) identified a family of 3′-co-terminal spliced transcripts that includes M73 and M73.5. The current study investigated whether similar families of spliced genes also exist in the human CMV (HCMV) and rat CMV (RCMV) genomes. Northern blot, RT-PCR and RACE-PCR analysis of HCMV transcripts showed that while mRNAs from HCMV UL73 and a putative UL73.5 homologue were spliced and 3′-co-terminal, they were not 5′-co-terminal. In contrast, the spliced RCMV R73 and R73.5 transcripts were arranged in a similar manner to those in MCMV and found to be both 5′ and 3′-co-terminal. In both the HCMV and RCMV genomes, additional non-coding spliced transcripts were found to originate from these regions. These results highlight that families of spliced transcripts coding for structural glycoproteins are likely to be a conserved feature of this region of betaherpesviral genomes.