Abstract
Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.
Highlights
The function of the CXXXC motifs in Prolyl 3-hydroxylase 1 (P3H1) and CRTAP has not been previously characterized
Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process
Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the ␣-subunit of prolyl 4-hydroxylase and lysyl hydroxylases
Summary
The function of the CXXXC motifs in P3H1 and CRTAP has not been previously characterized. The P4H complex hydroxylates proline residues in the Yaa position of collagen Gly-Xaa-Yaa sequences to 4(R)hydroxyproline using its 2-oxoglutarate- and iron-dependent dioxygenase domains [20, 21]. P3H1 forms a hetero-trimeric complex with CRTAP and CypB, and this complex has been shown to be a multifunctional complex that acts as prolyl 3-hydroxylase, peptidyl prolyl cis-trans isomerase, and molecular chaperone [27, 28] This complex hydroxylates proline residues in collagen Gly-Pro-4(R)-hydroxyproline sequences to 3(S)-hydroxyproline [28]. We synthesized a model peptide GCRALCG, whose sequence is located in the third position of the amino-terminal domain of P3H1 and which is well conserved in various species (Table 1) This peptide was used to test whether the CXXXC sequence behaved in a similar way to the CXXC sequence. The purified P3H1 complex, which is a biologically functional protein complex in the rER, was used in these enzyme assays to measure its enzyme activities in vitro
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