title: Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein.
abstract: Many secretory proteins are synthesized as proforms that become biologically active through a proteolytic cleavage in the trans-Golgi complex or at a later stage in the secretory pathway. Haptoglobin (Hp) is unusual in that it is cleaved in the endoplasmic reticulum before it enters the Golgi. Here, we present evidence that the recently discovered complement C1r-like protein (C1r-LP) mediates this cleavage. C1r-LP has not previously been shown to possess proteolytic activity, despite its homology to trypsin-like Ser proteinases. We demonstrate that coexpression of the proform of Hp (proHp) and C1r-LP in COS-1 cells effected cleavage of proHp in the endoplasmic reticulum. This cleavage depended on proteolytic activity of C1r-LP because mutation of the putative active-site Ser residue abolished the reaction. Furthermore, incubation of affinity-purified C1r-LP and proHp led to the cleavage of the latter protein. ProHp appeared to be cleaved at the expected site because substitution of Gly for Arg-161 blocked the reaction. C1r-LP showed specificity for proHp, in that it did not cleave the proform of complement C1s, a protein similar to Hp particularly around the cleavage site. C1r-LP accounts for at least part of the endogenous proHp-cleavage activity because suppression of the C1r-LP expression by RNA interference reduced the cleavage of proHp by up to 45% in the cells of a human hepatoma cell line (HepG2).
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, P.O. Box 582, S-751 23 Uppsala, Sweden. krzysztof.wicher@imbim.uu.se
authors.forename: Krzysztof B
authors.initials: KB
authors.lastname: Wicher
authors.forename: Erik
authors.initials: E
authors.lastname: Fries
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D015923:Complement C1r / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003173:Complement C1s / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006242:Haptoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D006242:Haptoglobins; D011498:Protein Precursors; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C042034:prohaptoglobin; D015923:Complement C1r; D003173:Complement C1s