Elsevier

Genomics

Volume 83, Issue 6, June 2004, Pages 1083-1093
Genomics

Identification and characterization of CPAMD8, a novel member of the complement 3/α2-macroglobulin family with a C-terminal Kazal domain

https://doi.org/10.1016/j.ygeno.2003.12.005Get rights and content

Abstract

We have identified and characterized a novel member of the complement 3/α2-macroglobulin (C3/α2M) family named CPAMD8 (complement 3 and pregnancy zone protein-like, α2-macroglobulin domain-containing 8). The gene maps to chromosome 19p13.2–p13.3 and spans approximately 130 kb. The gene partially overlaps with the protease-activated receptor-4 (PAR4) gene in the reverse orientation. The cDNA consists of 40 exons (∼6 kb) and encodes a protein of 1885 amino acids. Similar to other proteins in this family, CPAMD8 contains a signal sequence, an RXXR processing site, and a thioester motif. In addition, CPAMD8 has a Kazal-type serine proteinase inhibitor/follistatin-like domain at the C-terminus. The intact CPAMD8 protein generated by in vitro transcription and translation resolved as a single band of about 200 kDa on SDS–PAGE. RT-PCR and immunoblot assays showed that CPAMD8 is expressed in a number of human tissues, most abundantly in the kidney, brain, and testis and at lower levels in heart, liver, and small intestine. CPAMD8 is also expressed in several types of cells in culture, in which it is proteolytically processed into two chains of about 70 and 130 kDa. The Kazal domain of CPAMD8 binds to heparin, and subcellular fractionation shows that CPAMD8 is membrane associated via ionic interaction. In response to immune stimulants, CPAMD8 expression is markedly up-regulated in cells in culture. Thus, CPAMD8 may, like other members of the C3/α2M family, function in innate immunity but in a localized manner.

Section snippets

Identification and cloning of CPAMD8

Using the six known protein sequences of the C3/α2M family members, we performed a homologue search of the human genome database with the tBLASTn program. Seven additional putative C3/α2M-like genes were found in three genomic contigs on chromosomes 7, 12, and 19 (Table 1). Four putative genes on chromosome 12 are clustered with α2M and PZP. CPAMD8 is located on chromosome 19 (19p13.2–p13.3). A putative cDNA was predicted with GENESCAN, consists of 40 exons (∼6 kb), and has significant

Discussion

We report the cloning and characterization of a novel member of the C3/α2M family. CPAMD8 is highly conserved during evolution from zebrafish to human. CPAMD8 orthologues showing 65–86% amino acid identity are present in several species such as pig, cow, chicken, Fugu, zebrafish, and Xenopus. Interestingly, there is no evidence that CPAMD8 exists in rodents. The PAR4 gene, which overlaps with CPAMD8 in the human genome, has been found on mouse chromosome 8, but CPAMD8 is absent from that

Materials

Superscript one-step RT-PCR, Lipofectamine 2000, pSecTag2B vector, and Precast gels for SDS–PAGE were purchased from Invitrogen (Carlsbad, CA, USA). The PROTEIN Script II Linked Transcription:Translation kit was purchased form Ambion (Austin, TX, USA). The Redivue l-[35S]methionine, pGEX-4T-1 vector, glutathione Sepharose 4B, and ECL Western blotting detect reagent were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Centricon centrifugal filter devices were from Millipore

Acknowledgements

We thank the Kazusa DNA Research Institute for the partial cDNA clone of CPAMD8, Tristan Williams for help with mass spectrometry, Helene Fisher and Darek Kedra for advice, and Torgny Stigbrand for comments on the manuscript. This research was supported by a grant from the National Institutes of Health to E.E.

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    Sequence data from this article have been deposited with the NCBI Data Library under the Accession No. AY101765. The amino acid sequence of this protein can be accessed through the NCBI protein database under NCBI Accession No. AAM50084.

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