Role of basement membrane in tumor growth and metastasis.

The basement membrane is a thin extracellular matrix produced by epithelial and endothelial cells. It is biologically active for normal epithelial cell differentiation. Basement membrane promotes the growth of tumor cells in vitro and in vivo when coinjected. Laminin, the major biologically active component, also increases tumor growth and the malignant phenotype by promoting increased cell growth and protease activity. Using systematic peptide screening with synthetic peptides covering the entire laminin molecule, several active sites in laminin have been identified that regulate tumor growth and metastasis.

Cell type-specific differences in glycosaminoglycans modulate the biological activity of a heparin-binding peptide (RKRLQVQLSIRT) from the G domain of the laminin alpha1 chain.

AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha1 chain, has diverse biological activities with different cell types. The heparan sulfate side chains of syndecan-1 on human salivary gland cells were previously identified as the cell surface ligand for AG73. We used homologous peptides from the other laminin alpha-chains (A2G73-A5G73) to determine whether the bioactivity of the AG73 sequence is conserved. Human salivary gland cells and a mouse melanoma cell line (B16F10) both bind to the peptides, but cell attachment was inhibited by glycosaminoglycans, modified heparin, and sized heparin fragments in a cell type-specific manner. In other assays, AG73, but not the homologous peptides, inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and truncations. Fewer residues were critical for inhibiting branching morphogenesis (XKXLXVXXXIRT) than those required to inhibit B16F10 network formation on Matrigel (N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a beta-sheet with the N-terminal K (Lys(2)) and the C-terminal R (Arg(10)) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.

Characterization of Trichomonas vaginalis AP33 adhesin and cell surface interactive domains.

Adherence to host target cells is a critical step in establishing infection with the sexually transmitted pathogen Trichomonas vaginalis. Four parasite surface proteins mediating attachment to vaginal epithelial cells have been identified. One surface protein, termed AP33, was characterized further to identify domains interactive with previously generated antibodies and with host surface sites. N- and C-terminal deletion subclones were generated and tested for reactivity with both mAb and rabbit antiserum against AP33, and were also examined for their ability to bind to host cells. Surprisingly, the rabbit antiserum known to inhibit cytoadherence recognized an epitope(s) contained within 72 residues in the N-terminal half of the protein. However, the mAb epitope was immunoreactive with a 28-amino-acid region near the C-terminus. Subsequent mapping of this region with overlapping peptides identified a nine-amino-acid sequence reactive with the mAb. Equally surprising, two domains interactive with host cell surfaces were identified at distinct parts of AP33: one in the N-terminal half of the protein, and the other within 24 residues in the C-terminal third. Further analysis of the C-terminal binding domain revealed that a peptide representing this area could inhibit T. vaginalis cytoadherence by 40%.

Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence.

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T. vaginalis isolates. Analysis of full-length sequences determined that each contained a 930bp open reading frame encoding a protein of approximately 33,000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N-terminal protein sequencing established the presence of leader peptides. Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase alpha-subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host-parasite relationship.

Only two of the Trichomonas vaginalis triplet AP51 adhesins are regulated by iron.

The sexually transmitted parasite Trichomonas vaginalis cytoadheres to the vaginal epithelium, and four candidate trichomonad adhesins have been identified. One such protein, termed AP51, was characterized further. To do this, we studied a 1 kb cDNA clone (AP51.2) isolated from a phagemid expression library, which encoded a fusion protein of approximately 38 kDa that was immuno-crossreactive with anti-AP51 serum and retained functional adhesive properties. We performed 5'-PCR amplification to recover the missing 5' end in order to provide the complete cDNA sequence for the gene encoded by AP51.2 (ap51-2). Other PCR products revealed almost complete sequences for two additional ap51 genes, making AP51 a member of a multigene family of at least three distinct proteins and genes. The ap51-1 and ap51-3 genes each encoded for 407 amino acids while ap51-2 encoded 408 amino acids, and not unexpectedly, these genes had a high percent identity at the DNA and amino acid levels. Mapping confirmed the sequence distinctions and uniqueness of the three ap51 genes. Southern analysis using gene-specific probes revealed the single copy nature of each of the ap51 genes, all of which were present among the numerous agar clones of single trichomonads of the isolates tested. Importantly, Northern analysis showed transcriptional regulation by iron of only the ap51-1 and ap51-3 genes but not ap51-2, perhaps indicating the presence of two bona fide isoforms of the ap51 genes. The 3'-untranslated region of ap51-3 had a short poly (A) tail as well as the sequence motif AUUUA, which may relate to differential degradation of ap51-3 transcripts, in comparison to ap51-1 and ap51-2. Finally, the ap51 genes had partial homology to the beta-subunit of succinyl-CoA synthetase, reinforcing the idea that molecular mimicry may play a role in host parasitism by T. vaginalis.

Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence.

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno-crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full-length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701-base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 83 087 daltons, respectively. Comparison of the full-length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction-enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65-1 and ap65-2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of approximately 1.8 kb for both ap65-1 and ap65-2 genes in trichomonads grown only in high-iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.

Characterization of cDNAs encoding adhesin proteins involved in trichomonas vaginalis cytoadherence.

Trichomonas vaginalis cytoadherence is mediated by four adhesins (AP65, AP51, AP33 and AP23). Adhesin gene expression was previously shown to be up-regulated by iron. Therefore, a cDNA library was constructed from mRNA of T. vaginalis grown in a high-iron medium. Ten cDNA clones (three for AP65, one for AP51, and six for AP33) were recognized with polyclonal antiserum or monoclonal antibody raised against these adhesins. No cross-hybridization among cDNAs of the three adhesins was observed in Southern analysis, confirming restriction mapping analysis of representative cDNAs. Southern analysis probed with representative cDNAs of the adhesins indicated multiple copies for each of the three adhesin genes. Northern analysis showed transcripts of 1.8 kilobases (kb), 1.4 kb, and 0.9 kb for AP65, AP51 and AP33, respectively. Consistent with adhesin expression, mRNAs for these adhesins were detected only when parasites were grown in high-iron conditions. Specific antibodies eluted from E. coli expressing recombinant proteins reacted only with the respective parasite adhesins. Recombinant proteins bind to fixed HeLa cells and competed with radiolabeled trichomonad adhesins for binding. Although proteinase activity is required for cytoadherence, recombinant proteins show no detectable proteinase activity. These data show that recombinant proteins from these clones exhibited characteristics of the trichomonad adhesins.

Molecular basis of host epithelial cell recognition by Trichomonas vaginalis.

Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65,000 daltons (65 kDa; AP65), 51 kDa (AP51), 33 kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long-term-grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti-adhesin antibodies reacted by immunoblot only with the respective protein and detected, by indirect immunofluorescence, each adhesion on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact-dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesions also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.

Vaginal antibody of patients with trichomoniasis is to a prominent surface immunogen of Trichomonas vaginalis.

Twenty vaginal washes (VWs) and ten vaginal mucus (VM) samples from patients with trichomoniasis were examined for the presence of antibody to surface protein immunogens of Trichomonas vaginalis. Fourteen of 20 VWs (70%) and 8 of 10 VM (80%) had immunoglobulin G (IgG) antibody (Ab) that reacted in an immunoprecipitation (IP) assay with one iodinated Trichomonas vaginalis surface protein immunogen with a relative molecular mass of 230,000 daltons (230-kDa) (P230). No similar IP of any iodinated protein was observed when detergent extract was first depleted of P230 with monoclonal antibody (MAb), indicating a highly specific VW IgG response of patients to P230. VWs were also obtained from 10 patients from one to four weeks after treatment. These VWs had the same, or in one case a greater, level of IgG to P230. Under no circumstances was Ab to P230 or any other trichomonad protein detected in VWs or VM from normal, uninfected women. Flow cytofluorometry with VW Ab yielded heterogeneous fluorescent and non-fluorescent populations of trichomonads, reaffirming the restricted Ab response to one or a few epitopes on P230 in the vagina of patients. Under identical conditions, the MAb gave totally fluorescent parasite populations of some isolates, and the MAb again demonstrated variable epitope accessibility to Ab binding (Infect Immun 1987;55:1037). Finally, the MAb or VW Ab was never cytolytic for immunoreactive (fluorescent) parasites, even in the presence of complement. This study identifies the most important trichomonad surface immunogen on the basis of the vaginal Ab response, and data underscore the significance of immune evasion strategies of this sexually transmitted disease agent.