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Structure and Activity of Human Pancreasin, a Novel Tryptic Serine Peptidase Expressed Primarily by the Pancreas*

  • Vikash J. Bhagwandin
    Affiliations
    Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
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  • Leola W.-T. Hau
    Affiliations
    Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
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  • Jon Mallen-St. Clair
    Affiliations
    Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
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  • Paul J. Wolters
    Affiliations
    Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
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  • George H. Caughey
    Correspondence
    To whom correspondence should be addressed: Cardiovascular Research Institute, University of California at San Francisco, CA 94143-0911. Tel.: 415-476-9794; Fax: 415-476-9749
    Affiliations
    Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
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  • Author Footnotes
    * This work was supported by Grant HL-24136 from the National Institutes of Health, by Grant 9RT-0152 of the University of California Tobacco-Related Disease Research Program, and by the Research and Development Program of the Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) and.
Open AccessPublished:November 18, 2002DOI:https://doi.org/10.1074/jbc.M209353200
      In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term “pancreasin” because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and γ-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.
      EST
      expressed sequence tag
      NA
      nitroanilide
      DISP
      distal intestinal serine protease
      CHO
      Chinese hamster ovary
      HPAC
      human pancreatic adenocarcinoma
      RACE
      rapid amplification of cDNA ends
      UTR
      untranslated region
      PBS
      phosphate-buffered saline
      rhpancreasin
      recombinant human pancreasin
      Serine proteases are a fertile family of hydrolases using the side-chain hydroxyl group of a precisely positioned serine to attack the carbonyl carbon of a target peptide bond (

      Barrett, A. J., Rawlings, N. D., and Woessner, J. F. (eds) (1998) Handbook of Proteolytic Enzymes, pp. 3-232, Academic Press, New York

      ). Despite this shared enzymatic mechanism, serine proteases as a group exhibit a tremendous range of target specificity. However, some members of the family recognize and cleave a narrow range of target sequences and are limitedin vivo to hydrolysis of essentially one type of target. An example is enteropeptidase, which is highly specific for pancreatic trypsinogens. Some enzymes, like activated pancreatic trypsin itself, are comparatively omnivorous, hydrolyzing the peptide bond of a broad range of peptides and proteins at sites containing basic amino acids. Other serine proteases cleave targets after aromatic, neutral aliphatic, or acidic residues, but mammalian serine proteases with tryptic specificity are particularly numerous and variable in form and function. These include many familiar proteases with roles in digestion, hemostasis, fibrinolysis, and activation of complement (
      • Caughey G.H.
      ). One of the more intriguing subgroups of tryptic serine proteases includes prostasin (
      • Yu J.X.
      • Chao L.
      • Chao J.
      ,
      • Yu J.X.
      • Chao L.
      • Chao J.
      ,
      • Yu J.X.
      • Chao L.
      • Ward D.C.
      • Chao J.
      ), testisin (
      • Hooper J.D.
      • Nicol D.L.
      • Dickinson J.L.
      • Eyre H.J.
      • Scarman A.L.
      • Normyle J.F.
      • Stuttgen M.A.
      • Douglas M.L.
      • Loveland K.A.
      • Sutherland G.R.
      • Antalis T.M.
      ,
      • Hooper J.D.
      • Bowen N.
      • Marshall H.
      • Cullen L.M.
      • Sood R.
      • Daniels R.
      • Stuttgen M.A.
      • Normyle J.F.
      • Higgs D.R.
      • Kastner D.L.
      • Ogbourne S.M.
      • Pera M.F.
      • Jazwinska E.C.
      • Antalis T.M.
      ,
      • Honda A.
      • Yamagata K.
      • Sugiura S.
      • Watanabe K.
      • Baba T.
      ), and γ-tryptase (
      • Wong G.W.
      • Tang Y.
      • Feyfant E.
      • Sali A., Li, L., Li, Y.
      • Huang C.
      • Friend D.S.
      • Krilis S.A.
      • Stevens R.L.
      ,
      • Caughey G.H.
      • Raymond W.W.
      • Blount J.L.
      • Hau L.W.-T.
      • Pallaoro M.
      • Wolters P.J.
      • Verghese G.M.
      ). These enzymes are tryptic in specificity (i.e.prefer arginines and lysines in target peptides) and are synthesized with a distinctive carboxyl-terminal peptide or glycosylphosphatidyl inositol membrane anchor. Subsequently, they may be released from their anchor and secreted. The genes of these three enzymes share an idiosyncratic organization of introns and exons and reside on the short arm of chromosome 16 (5, 10, 11). However, they differ widely in dominant tissue pattern of expression: i.e. kidney and prostate (prostasin) (
      • Yu J.X.
      • Chao L.
      • Chao J.
      ,
      • Yu J.X.
      • Chao L.
      • Chao J.
      ,
      • Narikiyo T.
      • Kitamura K.
      • Adachi M.
      • Miyoshi T.
      • Iwashita K.
      • Shiraishi N.
      • Nonoguchi H.
      • Chen L.M.
      • Chai K.X.
      • Chao J.
      • Tomita K.
      ), eosinophils, testicular germ cells and sperm (testisin) (
      • Hooper J.D.
      • Nicol D.L.
      • Dickinson J.L.
      • Eyre H.J.
      • Scarman A.L.
      • Normyle J.F.
      • Stuttgen M.A.
      • Douglas M.L.
      • Loveland K.A.
      • Sutherland G.R.
      • Antalis T.M.
      ,
      • Honda A.
      • Yamagata K.
      • Sugiura S.
      • Watanabe K.
      • Baba T.
      ,
      • Inoue M.
      • Kanbe N.
      • Kurosawa M.
      • Kido H.
      ), and airway and gut mast cells (γ-tryptase) (
      • Wong G.W.
      • Tang Y.
      • Feyfant E.
      • Sali A., Li, L., Li, Y.
      • Huang C.
      • Friend D.S.
      • Krilis S.A.
      • Stevens R.L.
      ,
      • Caughey G.H.
      • Raymond W.W.
      • Blount J.L.
      • Hau L.W.-T.
      • Pallaoro M.
      • Wolters P.J.
      • Verghese G.M.
      ). The functions of these proteases are being actively investigated. In the case of prostasin, one likely role that has emerged is regulation of transmembrane ion flux via epithelial sodium channels (
      • Vuagniaux G.
      • Vallet V.
      • Jaeger N.F.
      • Pfister C.
      • Bens M.
      • Farman N.
      • Courtois-Coutry N.
      • Vandewalle A.
      • Rossier B.C.
      • Hummler E.
      ). This non-classic regulatory role for one member of the prostasin subgroup of tryptic mammalian serine proteases hints that we can expect unconventional roles for other members of the subgroup.
      This laboratory's interest in γ-tryptase and prostasin (
      • Caughey G.H.
      • Raymond W.W.
      • Blount J.L.
      • Hau L.W.-T.
      • Pallaoro M.
      • Wolters P.J.
      • Verghese G.M.
      ) led us to seek genes and transcripts encoding related enzymes in the human genome. As detailed below, our search identified a new family member, which we term “pancreasin” because it appears to be predominantly transcribed by pancreatic tissue as well as by a cell line derived from pancreatic ductal epithelium. The pancreasin gene shares the idiosyncratic gene structure of human prostasin, testisin, and γ-tryptase and resides like the others on chromosome 16p. Furthermore, recombinant expression reveals that it is a catalytically competent, tryptic peptidase, and proteinase. However, its substrate preferences and inhibitor profile are unique and, unlike its closest relatives, it is synthesized and secreted without a membrane anchor. The distinct patterns of expression, catalytic, and structural features predict that pancreasin's functions are distinct from those of its closest known relatives.

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