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

doi:10.1016/S0378-1119(01)00462-0

Gene

Volume 270, Issues 1–2, 30 May 2001, Pages 93-102

https://doi.org/10.1016/S0378-1119(01)00462-0 Get rights and content

Abstract

In this report we describe the discovery of Eppin (Epididymal protease inhibitor), a gene on human chromosome 20 expressing three mRNAs encoding two isoforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Eppin'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 transcription initiation sites are present for both of the different Eppin 5′ UTRs and examination of the promoter region 1800 bp upstream of the start codon revealed a number of putative transcription enhancer binding sites typical of genes expressed in the epididymis or testis. Northern blot and tissue specific PCR data indicate Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis. Antiserum prepared against recombinant EPPIN recognizes several strong bands on Western blots of human epididymal extracts from the caput and corpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejaculated spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-type 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.

Introduction

Spermatozoa undergo a variety of post-testicular changes in the epididymis to acquire both progressive motility and fertilizing ability (Bedford, 1967, Orgebin-Crist, 1967). Absorption and secretion by epididymal epithelia lead to a changing fluid environment (Hinton and Palladino, 1995) that surrounds spermatozoa during their epididymal transit, changing the spermatozoan membrane composition (Scott et al., 1967), including the modification, acquisition and removal of various surface glycoproteins (Cooper, 1995, Eddy and O'Brien, 1994, Legare et al., 1999, Robaire et al., 2000). Glycosyltransferases and glycosidases found in epididymal fluid are responsible for many of the sperm surface modifications (Eddy and O'Brien, 1994, Jones, 1998), while proteases, such as procathepsin L (Okamura et al., 1995), ACE and serine proteases also play roles in both sperm surface modifications and sperm maturation (He et al., 1995, Kirchhoff et al., 1997, Dacheux et al., 1998, Phelps et al., 1990). Concomitant with the expression of proteases is the expression of their inhibitors; α₂-macroglobu!in, cystatin C, cystatin-like proteins of the Cres gene family (Cornwall et al., 1992, Cornwall et al., 1999, Dacheux et al., 1998), and HE4 (Kirchhoff et al., 1991).

Proteases play key roles in many physiological processes and their regulation by inhibitors is important for maintaining homeostasis (Potempa et al., 1994). The presence of proteases in the epididymis requires the presence of their inhibitors as an important regulatory mechanism for regional maturation and processing of spermatozoa. To understand the regulatory mechanisms of epididymal function, a necessary first step is an understanding of the control of the genes responsible for the epididymal proteins found within the regional specializations and fluid contents. In this study we report the identification of a new serine protease inhibitor, EPPIN, characterized by both WAP-type and Kunitz-type consensus sequences. The three splice variants of Eppin are expressed differently; Eppin-1 is expressed in the testis and epididymis, Eppin-2 is expressed in the epididymis and Eppin-3 in the testis. These variants may represent the first members of a family of protease inhibitors characterized by dual inhibitor consensus sequences on human chromosome 20.

Section snippets

Materials

All oligonucleotides were synthesized at the UNC-CH Nucleic Acids Core Facility. cDNAs were produced with the Superscript Kit (Life Technologies, Gaithersburg, MD). Purifications of plasmid and PCR DNAs were performed using the respective kits from Qiagen (Valencia, CA). The Multiple Tissue Northern Blot and Multiple Tissue cDNA Panel were purchased from Clontech, Inc. (Palo Alto, CA). Miscellaneous chemicals of the highest possible grade were purchased from Sigma (St. Louis, MO). Human

Cloning of the human Eppin cDNAs

The initial Eppin cDNA clone from the Human Genome Sciences database contained a 741 bp insert with an open reading frame (ORF) encoding a 133 amino acid protein with a deduced molecular weight of 15,283 Da and a predicted pI of 8.03 (Eppin-1, Fig. 1). Sequence analysis of the EPPIN-1 protein indicated two different protease inhibitor consensus sequences; (1) the four disuffide core type, C-x-{C}-[DN]-x(2)-C-x(S)-C-C, at positions 48–61 (shown by a double underline, Eppin-1, Fig. 1) and (2) the

Discussion

Eppin is a gene on human chromosome 20 characterized by three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. Analysis of Eppin'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 (Fig. 1, Fig. 2; Table 1). Eppin would appear to be a single copy gene (Fig. 3) and TATA box

Acknowledgements

Support for this project was provided by the CICCR Program of CONRAD [CIG-96-06-A], NIH grant U54HD29099 through the Center for Recombinant Gamete Vaccinogens, University of Virginia and by NICHD/NIH through cooperative agreement U54HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research. The authors thank Gail Grossman and the Immunohistochemistry Core Facility of the Laboratories for Reproductive Biology for their help and expertise.

References (37)

N Okamura et al.
Direct evidence for the elevated synthesis and secretion of procathepsin L in the distal caput epididymis of boar
Biochim. Biophys. Acta
(1995)
J Potempa et al.
The serpin superfamily of proteinase inhibitors: structure, function, and regulation
J. Biol. Chem.
(1994)
R Richardson et al.
Cloning and sequencing of cDNAs for rabbit preproacrosin and a novel preproacrosin-related cDNA
Dev. Biol.
(1994)
J Tan et al.
Response elements of the androgen-regulated C3 gene
J. Biol. Chem.
(1992)
M Ulvsback et al.
Gene structure of seminogelin I and II. The predominant proteins in human semen are encoded by two homologous genes on chromosome 20
J. Biol. Chem.
(1992)
J.L Workman et al.
Binding of transcription factor TFIID to the major late promoter during in vitro nucleosome assembly potentiates subsequent initiation by RNA polymerase II
Cell
(1987)
J.M Bedford
Effects of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis
J. Exp. Zool.
(1967)
A Bjartell et al.
Distribution and tissue expression of semenogelin I and II in man as demonstrated by in situ hybridization and immunocytochemistry
J. Androl.
(1996)
J.M Chirgwin et al.
Isolation of biochemically active ribonucleic acid from sources enriched in ribonuclease
Biochem. J.
(1979)
T.G Cooper
Role of the epididymis in mediating changes in the male gamete during maturation
Adv. Exp. Med. Biol.
(1995)

G.A Cornwall et al.

The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis

Mol. Endocrinol.

(1992)

G.A Cornwall et al.

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

Biochem. J.

(1999)

J.-L Dacheux et al.

Role of epididymal secretory proteins in sperm maturation with particular reference to the boar

J. Reprod. Fert. Suppl.

(1998)

J.R Drevet et al.

The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5

Mol. Reprod. Dev.

(1998)

E.M Eddy et al.

The Spermatozoon

U.K Ewulonu et al.

Promoter mapping of the Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences

Mol. Reprod. Dev.

(1996)

K.G Hamil et al.

HE2β and HE2γ, new members of an epididymis-specific family of androgen-regulated proteins in the human

Endocrinology

(2000)

X He et al.

The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues

J. Histochem. Cytochem.

(1995)

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

Abstract

Introduction

Section snippets

Materials

Cloning of the human Eppin cDNAs

Discussion

Acknowledgements

Biochim. Biophys. Acta

J. Biol. Chem.

Dev. Biol.

J. Biol. Chem.

J. Biol. Chem.

Cell

Effects of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis

J. Exp. Zool.

Distribution and tissue expression of semenogelin I and II in man as demonstrated by in situ hybridization and immunocytochemistry

J. Androl.

Isolation of biochemically active ribonucleic acid from sources enriched in ribonuclease

Biochem. J.

Role of the epididymis in mediating changes in the male gamete during maturation

Adv. Exp. Med. Biol.

The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis

Mol. Endocrinol.

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

Biochem. J.

Role of epididymal secretory proteins in sperm maturation with particular reference to the boar

J. Reprod. Fert. Suppl.

The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5

Mol. Reprod. Dev.

The Spermatozoon

Promoter mapping of the Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences

Mol. Reprod. Dev.

HE2β and HE2γ, new members of an epididymis-specific family of androgen-regulated proteins in the human

Endocrinology

The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues

J. Histochem. Cytochem.