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 affinitypurified 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).

Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

“…16 Automated Edman degradation was performed by using an Applied Biosystems Model 477A sequencer (Applied Biosystems, Foster City, CA) with on-line phenylthiohydantoin analysis by high-performance liquid chromatography (HPLC; Model 120A; Applied Biosystems). In agreement with recent data demonstrating a very low degree of cleavage of recombinant Hp to ␣-and ␤-chains in 293 cells, 17 amino-terminal sequencing of recombinant Hp yielded only the sequence of the mature ␣-chain amino-terminus (VDSGNDVTDIAD). Two aminoterminal sequences in equimolar amounts were obtained for recombinant Hpr (LYSGNDVTDISD and ILGGHLDAKGSF).…”

A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex

“…In accordance with recent data showing that only a low degree of cleavage of recombinant proHp to the ␣-and ␤-chains takes place in 293 cells (19), amino-terminal sequencing showed that recombinant Hp remained largely uncleaved (14). However, the recombinant Hp bound to Hb has similar receptor binding activity as Hb in complex with native plasmaderived Hp (14).…”

Haptoglobin, a hemoglobin-binding plasma protein, is present in bony fish and mammals but not in frog and chicken

“…In contrast, the primary translation product of mammalian Hp mRNA consists of a signal sequence, a CCP, and a SP domain. After the release of the signal sequence and while still in the endoplasmic reticulum, this polypeptide is cleaved between the two domains by C1r-like protein (19). Whether the difference in cleavage mechanisms between HpL and Hp has any physiologic significance remains to be studied.…”