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).

Fig. 1.

Detection of proHp cleaving activity in human serum and in the material from the last two steps of a commercial production of an albumin solution. Medium from cells expressing proHp and labeled with [³⁵S]Met (12 μl) was mixed with the sample to be analyzed (1 μl) and incubated at 37°C for 30 min. After immunoprecipitation and SDS/PAGE, radiolabeled Hp was detected by PhosphorImaging. The samples analyzed were buffer (lane 1), human serum (lane 2), material from the second-to-last step in the preparation of a commercial albumin solution (lane 3), and from the final, heat-treated albumin solution (lane 4); the protein concentrations were 75-150 mg/ml. PHp, α,and β, mark the positions of the proform, and the α- and β-chains of Hp, respectively. The molecular masses (in kilodaltons) of reference proteins are shown on the left.

Fig. 2.

Analysis by 2D gel electrophoresis of the fraction with the highest proHp cleaving activity obtained in the final step of purification starting from the albumin solution described in Fig. 1 (lane 3). Proteins were separated by isoelectric focusing in a pH gradient of 3-6, followed by SDS/12.5% PAGE. Shown is the part of the gel that contained proteins visualized by staining with Coomassie brilliant blue. Protein spots were excised, treated with trypsin, and analyzed by MS. Spots marked with numbers produced high Mowse scores for the corresponding proteins: 1, 2, complement C1r-like protein (GenBank accession no. gi|7706083|); 3, acid sphingomyelinase-like phosphodiesterase 3a (GenBank accession no. gi|18202617|); 4, 5, 6, soluble IL-1 receptor accessory protein-beta (GenBank accession no. gi|33286873|); 7, 8, 9, 10, leucine-rich α₂-glycoprotein (GenBank accession no. gi|72059|); 11, 12, 13, Zn-α₂-glycoprotein chain B (GenBank accession no. gi|4699583|); and 14, a protein similar to the 40 S ribosomal protein S17 (GenBank accession no. gi|20554971|). Those spots marked with an asterisk did not produce any hit in the database search. The molecular masses (in kilodaltons) of reference proteins are shown on the left.

Fig. 3.

Cleavage of proHp by recombinant C1r-LP. (A) Cells were transfected with vector expressing myc-His tagged C1r-LP or mock vector, and cells and media were analyzed by Western blotting with antibodies to the myc epitope. The molecular masses (in kilodaltons) of reference proteins are shown on the right. (B) Cells were transfected with vector expressing proHp, and either mock vector, or vector expressing either C1r-LP, or a C1r-LP S⁴³⁶A mutant (C1r-LP^*). Subsequently, cell media were analyzed by Western blotting with antibodies to Hp (α-Hp) or to myc-tag (α-myc). (C) myc-His₆-tagged proHp and C1r-LP were isolated as described in Materials and Methods. ProHp was mixed with C1r-LP or buffer only and incubated for 16 h at 37°C. Subsequently, proHp cleavage was analyzed by Western blotting.

Fig. 4.

Time course of intracellular cleavage of proHp by C1r-LP. (A) COS-1 cells expressing proHp and C1r-LP were pulse-labeled for 5 or 7.5 min with [³⁵S]Met and chased for the indicated times. Cells were then solubilized and subjected to immunoprecipitation with antibodies to Hp. After SDS/PAGE, radiolabeled Hp was visualized by PhosphorImaging. Only the top part of the gel is shown. PHp and β mark the positions of the proform and the β-chain of Hp, respectively. (B) Some of the samples described in A, as well as the medium obtained after 240 min of chase, were treated with EndoH before electrophoresis. PHp, β_c, and β_m mark the positions of the proform, cellular β-chain, and (Endo H-resistant) extracellular β-chain of Hp, respectively. PHp^Δ and indicate the respective deglycosylated forms appearing on Endo H treatment.

Fig. 5.

Specificity of proHp cleavage by C1r-LP. (A) Alignment of amino acid sequences around cleavage site in proHp, proC1s, and proC1r. For comparison, the corresponding sequence of C1r-LP is also shown. Identical amino acid residues are bold and the position of the cleavage site is marked with an arrowhead. (B) Media of COS-1 cells expressing proC1s alone, or proC1s with either C1r-LP or proC1r were analyzed by Western blotting with antibodies to C1s (α-C1s). pC1s, A, and B mark the positions of proC1s, and the A and B chains of C1s, respectively. Expression of C1r-LP was assessed by Western blotting with antibodies to the myc epitope (α-myc). (C) Media of cells transfected with vector expressing proHp, and either mock vector, or vector expressing C1r were analyzed by Western blotting with antibodies to Hp. pHp, α, and β mark the positions of proHp, and the α- and β-chains of Hp, respectively. (D) Cells were transfected with vector expressing a proHp R-161G mutant, and either mock vector, or vector expressing C1r-LP. Subsequently, cell media were analyzed by Western blotting with antibodies to Hp (α-Hp). pHp^*, α, and β mark the positions of the mutated proform, and the α- and β-chains of Hp, respectively. Expression of C1r-LP was assessed by Western blotting with antibodies to the myc epitope (α-myc).

Fig. 6.

Inhibition of proHp cleavage by siRNA specific for C1r-LP. (A) HepG2 cells were transfected with 50 nM siRNA specific for either C1r-LP, C1r, or irrelevant siRNA, as described in Materials and Methods. Subsequently, cell media were analyzed by Western blotting with antibodies to Hp. (B) HEK293 cells were transiently transfected with vector expressing proHp, and vector expressing either irrelevant siRNA, C1r-LP siRNA, or C1r-LP. Cell extracts were then analyzed by Western blotting with antibodies to Hp.