The WFDC1 gene: role in wound response and tissue homoeostasis

The present evaluates the key features of the WFDC1 [WAP (whey acidic protein) four disulfide core 1] gene that encodes ps20 (20 kDa prostate stromal protein), a member of the WAP family. ps20 was first characterized as a growth inhibitory activity that was secreted by fetal urogenital sinus mesenchymal cells. Purified ps20 exhibited several activities that centre on cell adhesion, migration and proliferation. The WFDC1 gene was cloned, contained seven exons, and was mapped to chromosome 16q24, suggesting that it may function as a tumour suppressor; however, direct evidence of this has not emerged. In vivo, ps20 stimulated angiogenesis, although expression of WFDC1/ps20 was down-regulated in the reactive stroma tumour microenvironment in prostate cancer. WFDC1 expression is differential in other cancers and inflammatory conditions. Recent studies point to a role in viral infectivity. Although mechanisms of action are not fully understood, WFDC1/ps20 is emerging as a secreted matricellular protein that probably affects response to micro-organisms and tissue repair homoeostasis.     

Cathepsin-L and transglutaminase dependent processing of ps20: A novel mechanism for ps20 regulation via ECM cross-linking

Whey-acidic-protein (WAP) four-disulphide core (WFDC) proteins have important roles in the regulation of innate immunity, anti-microbial function, and the inhibition of inflammatory proteases at mucosal surfaces. It was recently demonstrated that the WFDC protein, prostate stromal 20 (ps20), encoded by the WFDC1 gene, is a potent growth inhibitory factor, and shares with other WFDC proteins the ability to modulate wound healing processes and immune responses to viral infections. However, ps20 remains relatively uncharacterised at the protein level.Using a panel of ps20 antibodies for western-blotting (WB), ELISA and immunoaffinity purification, we isolated, biochemically characterised and tested ps20 preparations for three biological properties: (i) interactions with glycosaminoglycans (GAG) (ii) inhibition of cell proliferation, and (iii) transglutaminase2 (TG2) mediated crosslinking of ps20 to fibronectin, a process implicated in wound healing. We show herein that ps20 preparations contain multiple molecular forms including full-length ps20 (resolving at ≈27 kDa), an exon 3 truncated form (≈22 kDa) that lacks aa113–140, and variable amounts of a putatively cleaved lower MW (≈15–17 kDa) species. Untagged purified ps20 preparations containing a mixture of these forms are biologically active in significantly suppressing prostate cell proliferation. We show that one mechanism by which lower LMW forms of ps20 arise is through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. However, CL cleavage abrogated the interaction between ps20 and solid-phase fibronectin.Therefore, we demonstrate for the first time that LMW forms of ps20 that lack a C-terminal immunogenic epitope can arise through CL cleavage and this cleavage impairs multimerisation and potential capacity to cross-link to ECM, but not the capacity of ps20 to inhibit cell proliferation. We propose that ps20 like other WFDC proteins can become associated with GAGs and the ECM. Furthermore, we suggest post-translational processing and cleavage of ps20 is required to generate functional protein species, and TG2 mediated crosslinking and CL cleavage form components of a ps20 regulatory apparatus.     

The evolution of a genetic locus encoding small serine proteinase inhibitors

We previously identified a locus on human chromosome 20 that encompasses 14 genes of postulated WFDC-type proteinase inhibitors with a potential role in innate immunity. In an extended study, homologous loci are here described on mouse chromosome 2, rat chromosome 3, and dog chromosome 24. As in humans, the murine and canine loci are divided into two sub-loci separated by 0.2Mb. The majority of genes are conserved in all species, but there are also species-specific gains and losses of genes, e.g., several duplications have yielded four SLPI genes in the rat and, most surprisingly, there is no murine elafin gene. Two human pseudogenes were identified due to the discovery of functional rodent genes. The conservation of different WFDC domains varies considerably, and it is hypothesized that this reflects a dual role of WFDC inhibitors in natural immunity, which is directed both against microbes and proinflammatory cells.     

Murine phosphatidylserine-specific phospholipase A1 (Ps-pla1) maps to Chromosome 16 but is distinct from the lpd (lipid defect) locus

We have previously generated a mouse transgenic line with an insertional mutation designated lpd that demonstrates a phenotype of hypertriglyceridemia and fatty liver. Since the recently identified phosphatidylserine-specific phospholipase A1 (PS-PLA1) demonstrates significant homology to triglyceride lipases, we reasoned that the mouse Ps-pla1 gene may be the disrupted gene within the lpd locus. Using a rat PS-PLA1 cDNA sequence to search the EST database, we identified a mouse EST homolog AA839424. Sequencing analysis of AA839424 revealed a putative Ps-pla1 protein of 456 amino acids with extensive overall structural conservation with human and rat PS-PLA1 and with triglyceride lipases. Conserved sequences in Ps-pla1 include a lipase consensus sequences G×S×G, a catalytic triad, and eight of the ten conserved cysteine residues that are required for tertiary structure. Mouse Ps-pla1 carries a phosphatidylserine-binding motif that is absent in all triglyceride lipases. Using a mouse whole-genome radiation hybrid (WG-RH) mapping panel (T31), we mapped mouse Ps-pla1 to Chromosome (Chr) 16 between genetic markers D16Mit194 and D16Mit38, which is 17.1 cM centromeric to the lpd locus. On the basis of chromosome location, we conclude that Ps-pla1 and lpd are distinct genes in lipid metabolism. Received: 31 May 2000 / Accepted: 5 October 2000     

Structural Organization of the Mouse and Human GALR1 Galanin Receptor Genes (GalnrandGALNR) and Chromosomal Localization of the Mouse Gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15–20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouseGalnrgene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the humanGALNRgene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Characterization of a Human Glycoprotein with a Potential Role in Sperm–Egg Fusion: cDNA Cloning, Immunohistochemical Localization, and Chromosomal Assignment of the Gene (AEGL1)

Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that noAEGmRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescencein situhybridization to 6p21.1–p21.2. This result indicates thatAEGL1and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies.     

Genes encoding WFDC- and Kunitz-type protease inhibitor domains: are they related?

We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).     

The ras-related ypt protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPT1 gene.

The YPT1 gene of the yeast Saccharomyces cerevisiae codes for a guanine nucleotide-binding protein which is essential for cell viability. Using as hybridization probe cloned yeast YPT1 gene sequences, we have isolated from cDNA libraries prepared from RNA of mouse F9 and C3H10T1/2 cells several overlapping cDNA clones with identical sequence in the regions of overlap. The cDNAs were derived from a gene, designated ypt1, which codes for a protein of 205 amino acids with 71% homology to the yeast YPT1 gene product. Amino acid sequences typical for guanine nucleotide-binding proteins and characteristic for ypt proteins are perfectly conserved in the mouse ypt1 protein. Two mRNAs of 1600 and 3200 nucleotides, originating from the mouse ypt1 gene and differing in the length of their 3'-non-translated region, were identified in mouse F9 cells and in all mouse tissues examined. A monoclonal antibody specifically recognizing the 23.5-kd yeast YPT1 protein cross-reacted with a protein of identical size on protein blots of mouse, rat, pig, bovine and human cell lines.     

Mouse and Human Neuronal Pentraxin 1 (NPTX1): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1–q25.2 and chromosome 11e2–e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.     

Identification and characterization of the mouse MutS homolog 5: Msh5

We have identified and characterized the complete cDNA and gene for the mouse MutS homolog 5 (Msh5), as a step toward understanding the molecular genetic mechanisms involved in the biological function of this new MutS homologous protein in mammals. The Msh5 cDNA contains a 2502-bp open reading frame (ORF) that encodes an 833-amino acid protein with a predicted molecular weight of 92.6 kDa, which shares 89.8% amino acid sequence identity with the human hMSH5 protein. Northern blot analysis demonstrated the presence of a Msh5 mRNA approximately 2.9-kb in length, most abundantly expressed in mouse testis. Yeast two-hybrid analysis indicated that the mouse Msh5 protein positively interacted with the human hMSH4 protein—suggesting that Msh5 shares common functional properties with its human counterpart. Sequence and structural analyses show that the mouse gene Msh5 spans approximately 18 kb and contains 24 exons that range in length from 36 bp for exon 7 to 392 bp for exon 1. Structural comparison with the human hMSH5 gene revealed that all of the Msh5 internal exons, but not introns, are conserved in length with the human hMSH5. The Msh5 gene is located on mouse Chromosome (Chr) 17 in a location that is syntenic to the region of human Chr 6 harboring the hMSH5 gene. The identification and characterization of Msh5 will facilitate studies of the potential functional roles of this new member of the MutS family. Received: 11 May 1999 / Accepted: 16 July 1999     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998