Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor ␣ subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-␣ ODDD reporter construct levels, and its small interfering RNA increased HIF-1␣ protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1␣ ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.Prolyl 4-hydroxylases (P4Hs) 2 catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in peptide linkages. Two animal P4H families are known today: collagen P4Hs (C-P4Hs), endoplasmic reticulum (ER) luminal enzymes that have a central role in the synthesis of all collagens (1-3), and HIF-P4Hs, nuclear and cytoplasmic enzymes that play a key role in the response of cells to hypoxia (4 -7).The C-P4Hs act on Xaa-Pro-Gly sequences in collagens and more than 20 collagen-like proteins, the 4-hydroxyproline residues formed being essential for the assembly of triple-helical molecules (1-3). All vertebrate C-P4Hs are ␣ 2 ␤ 2 tetramers in which the enzyme and chaperone protein disulfide-isomerase (PDI) acts as the ␤ subunit (1-3). Three isoforms of the catalytic ␣ subunit have been cloned and characterized and found to form [␣(I)] 2 ␤ 2 , [␣(II)] 2 ␤ 2 , and [␣(III)] 2 ␤ 2 tetramers with PDI, known as C-P4Hs I, II, and III, respectively (3,8,9).The HIF-P4Hs regulate the hypoxia-inducible factors (HIFs) by hydroxylating proline residues in Leu-Xaa-Xaa-Leu-Ala-Pro sequences at two separate sites in their ␣ subunits (10, 11). The human HIF-P4Hs have three isoenzymes, HIF-P4Hs 1-3 (also known as PHDs 1-3, HPHs 3-1, and EGLNs 2, 1, and 3, respectively), which show a 42-59% sequence identity to each other but essentially no sequence similarity to the C-P4Hs except for the catalytically critical residues (12-14). HIFs are ␣␤ heterodimers that act as master regulators of the transcription of more than 100 hypoxia-regulated genes (5-7). The human HIF-␣ subunit has three isoforms, HIF-1␣-HIF-3␣. HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of at least one of two critical proline residues in their oxygendependent degradation domain (ODDD), Pro 402 and Pro 564 in HIF-1␣, generates a binding site for the von Hippel-Lindau E3 ubiquitin ligase complex that tar...

4-Hydroxyproline is found in collagens, collagen-like proteins, elastin, and the hypoxia-inducible transcription factor in animals and in many hydroxyproline-rich glycoproteins in plants. We report here on the cloning and characterization of a second plant P4H (prolyl 4-hydroxylase), At-P4H-2, from Arabidopsis thaliana. It consists of 299 amino acids and shows 33% sequence identity to the first characterized isoenzyme, At-P4H-1. A characteristic feature of the At-P4H-2 polypeptide is a 49-amino-acid C-terminal toxin homology domain with 6 cysteines that is not found in At-P4H-1 but is present in a putative rice P4H homologue. At-P4H-2 differed distinctly from At-P4H-1 in its substrate specificity. Recombinant At-P4H-2 hydroxylated poly(L-proline) and extensin and arabinogalactan-like peptides effectively but with much higher K m values than At-P4H-1, suggesting different roles for the two At-P4Hs in the plant cell. Unlike At-P4H-1, At-P4H-2 hydroxylated collagen-like peptides only very inefficiently and did not hydroxylate hypoxia-inducible transcription factor ␣-like peptides at all. All the peptides efficiently hydroxylated by At-P4H-2 had at least 3 consecutive prolines, suggesting that these may represent a minimum requirement for efficient hydroxylation by this isoenzyme. N-terminal sequencing of an extensin-like peptide SPPPVYKSPPP-PVKHYSPPPV indicated that At-P4H-2 preferentially hydroxylated the 3rd proline in the C-terminal PPP triplet. The K m values of At-P4H-2 for the reaction cosubstrates Fe 2؉ , 2-oxoglutarate, and ascorbate were similar to those of At-P4H-1 with the exception that the K m for iron was about 3-fold lower. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate, well known competitive inhibitors of the vertebrate P4Hs with respect to 2-oxoglutarate, were also competitive inhibitors of At-P4H-2 but with K i values 5-100-fold higher than those of human type I collagen P4H. It thus seems that there are some distinct differences in the structure of the 2-oxoglutarate-binding site between At-P4H-2 and the animal collagen P4Hs.4-Hydroxyproline is found in animal proteins almost exclusively in collagens, elastin, and more than 20 additional proteins with collagen-like sequences (for reviews, see Refs.

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp) 5 as a substrate. The K m and K i values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.Collagen synthesis involves many unusual co-translational and post-translational modifications, including the formation of 4-hydroxyproline (4Hyp), 2 3-hydroxyproline, and hydroxylysine in -X-Pro-Gly-, -Pro-4Hyp-Gly-, and -X-Lys-Glysequences, respectively. These reactions take place within the lumen of the endoplasmic reticulum and are catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.

Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.