Identification and Characterization of a Third Human, Rat, and Mouse Collagen Prolyl 4-Hydroxylase Isoenzyme

Abstract

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of -X-Pro-Gly-triplets. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers, the ␤-subunit being identical to protein-disulfide isomerase (PDI). Two isoforms of the catalytic ␣-subunit, which combine with PDI to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, have been known up to now. We report here on the cloning and characterization of a third vertebrate C-P4H ␣-subunit isoform, ␣(III). The processed human, rat and mouse ␣(III) polypeptides consist of 520 -525 residues, all three having signal peptides of 19 -22 additional residues. The sequence of the processed human ␣(III) polypeptide is 35-37% identical to those of human ␣(I) and ␣(II), the highest identity being found within the catalytically important C-terminal region and all five critical residues at the cosubstrate binding sites being conserved. The sequence within a region corresponding to the peptide-substrate binding domain is less conserved, but all five ␣ helices constituting this domain can be predicted to be located in identical positions in ␣(I), ␣(II), and ␣(III) and to have essentially identical lengths. The ␣(III) mRNA is expressed in many human tissues, but at much lower levels than the ␣(I) and ␣(II) mRNAs. In contrast to ␣(I) and ␣(II), no evidence was found for alternative splicing of the ␣(III) transcripts. Coexpression of a recombinant human ␣(III) polypeptide with PDI in human embryonic kidney cells led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [␣(III)] 2 ␤ 2 tetramer. The catalytic properties of the recombinant enzyme were very similar to those of the type I and II C-P4Hs, with the exception that its peptide binding properties were intermediate between those of the type I and type II enzymes.

Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

“…Enzyme Activity Assay-The P4H activity of the Triton X-100-soluble insect cell fractions was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-14 C]glutarate (24), using a synthetic peptide (PPG) 10 (Peptide Institute) as the substrate. K m values for (PPG) 10 were determined by varying its concentration while the concentrations of the other reaction components were kept constant.…”

“…K m values for (PPG) 10 were determined by varying its concentration while the concentrations of the other reaction components were kept constant.…”

Osteocalcin protein sequences of Neanderthals and modern primates

“…P4Hs (EC 1.14.11.2) are discriminated by their protein substrate preferences, K m and kinetic properties, and subcellular localization. P4Hs all require molecular O 2 , L-ascorbic acid, 2-oxoglutarate, and Fe 2ϩ to catalyze the hydroxylation of Pro in peptides (15,16). Collagen P4Hs (Col-P4Hs) act in the endoplasmic reticulum to modify nascent procollagen chains.…”

“…The peptide substrate specificities for the HIF-P4Hs (Leu-X-X-Leu-Ala-Pro*-Ala͞Tyr) (16,19) suggest that they may hydroxylate Pro-9 in osteocalcin (Leu-Gly-Ala-Pro*-Ala͞Val) (Fig. 1) more efficiently than the Col-P4Hs, which target the Gly-X-Pro*-Gly sequence (15). Both P4H classes are expressed in osteoblasts where osteocalcin and collagen type I are synthesized, but the subcellular localization of osteocalcin hydroxylation has not been established.…”

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

“…The subunit contains the catalytic site with conserved residues required for the binding of the Fe 2+ and 2-oxoglutarate (4,5) and the peptide-substrate-binding domain (6,7). Three subunit isoforms have been characterized from human and mouse that assemble into [ (I)] 2 2 , [ (II)] 2 2 and [ (III)] 2 2 tetramers (8-12) and display distinct tissue distributions (11,13,14). The Drosophila melanogaster genome contains about 20 genes encoding collagen P4H subunit-like polypeptides, the one characterized in detail also assembling into an active 2 2 tetramer (15,16).…”