Cloning and sequencing of human Eppin: A novel family of protease inhibitors expressed in the epididymis and testis

In this report we describe the discovery of Eppin (Epididymal protease inhi bitor), a gene on human chromosome 20 expressing three mRNAs encoding two i soforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Epp in's genomic sequence from chromosome 20q12-13.2 predicts the existence of all three splice variants of Eppin and that all the exons conform to the AG /GT splicing rule. The presence of single bands on a Southern blot of human genomic DNA suggests that Eppin is a single copy gene. TATA box transcript ion initiation sites are present for both of the different Eppin 5 ' UTRs a nd examination of the promoter region 1800 bp upstream of the start codon r evealed a number of putative transcription enhancer binding sites typical o f genes expressed in the epididymis or testis. Northern blot and tissue spe cific PCR data indicate Egpin-1 is expressed only in the testis and epididy mis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the te stis. Antiserum prepared against recombinant EPPIN recognizes several stron g bands on Western blots of human epididymal extracts from the caput and co rpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejacula ted spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-ty pe and Kunitz-type consensus sequences. A second family member is predicted to exist on chromosome 20 approximately 4 kb downstream from Eppin's exon I, which has two WAP-type sequences and one Kumtz-type consensus sequence. (C) 2001 Elsevier Science B.V. All rights reserved.