Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase

Merriweather, Anthony; Guenzler, Volkmar; Brenner, Mary E.; Unnasch, Thomas R.

doi:10.1016/s0166-6851(01)00317-6

Cited by 17 publications

(14 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). The amino acid sequence homology between the B. malayi and C. elegans PHY polypeptides is slightly higher than that between the B. malayi PHY-1 and the PHY-1 from a closely related filarial nematode, Onchocerca volvulus (22), with the B. malayi and O. volvulus PHY-1 polypeptides being 49% identical and 70% similar. The amino acid sequence identities between the B. malayi PHY-1 and the human ␣(I) and ␣(II) subunits are 45 and 44%, and the similarities are 62 and 63%, respectively.…”

Section: Cloning Of B Malayi Phy-1-threementioning

confidence: 78%

“…Chemical inhibitors of P4H have also been shown to have similar cuticle-specific effects in both C. elegans (14,16) and B. malayi (22). As P4H is involved in the synthesis of the nematode cuticle, it is therefore an excellent target for parasite control by chemical inhibition.…”

Section: Fig 8 Developmental Expression Analysis Of B Malayi Phy-1mentioning

confidence: 99%

“…Lymphatic filariasis is a debilitating disease, with approximately onethird of those infected being incapacitated and/or disfigured by the infection (21). Commercially available inhibitors of P4H have been shown to be toxic to B. malayi adults, producing associated cuticular defects (22). These observations and the requirement in C. elegans for P4H activity highlight this enzyme class as a potential drug target in the control of human and veterinary parasitic nematode infections.…”

mentioning

confidence: 99%

See 2 more Smart Citations

A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase

Winter

Myllyharju

Page

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in the XPro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are ␣ 2 ␤ 2 tetramers with the catalytic activity residing in the ␣ subunits. The ␤ subunits are identical to the enzyme protein disulfide isomerase. The nematode cuticle is a collagenous extracellular matrix required for maintenance of the worm body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia malayi is a causative agent of lymphatic filariasis in humans. We report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi ␣ subunit, PHY-1, is a soluble and active P4H by itself, and it does not become associated with protein disulfide isomerase. The active enzyme form is a homotetramer with catalytic and inhibition properties similar to those of the C. elegans P4Hs. High levels of B. malayi phy-1 transcript expression were observed in all developmental stages examined, and its expression was localized to the cuticle-synthesizing hypodermal tissue in the heterologous host C. elegans. Although active by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant.Biosynthesis of vertebrate collagens requires processing by up to eight specific intra-and extracellular posttranslational enzymes (1). The collagen prolyl 4-hydroxylase (P4H) 1 class of enzymes (EC 1.14.11.2) catalyze the hydroxylation of prolines in the X-Pro-Gly repeats of collagen chains. This endoplasmic reticulum (ER) resident enzyme is central to collagen synthesis, as collagen triple helices are thermally unstable in the absence of 4-hydroxyproline residues (2, 3). P4H also acts as a chaperone in the assembly of collagen ensuring that only correctly folded collagens are released for secretion (4). In vertebrates and Drosophila melanogaster the enzyme is an ␣ 2 ␤ 2 tetramer (2, 3, 5, 6), with hydroxylation activity residing in the catalytic ␣ subunits. Two ␣ subunit isoforms, ␣(I) and ␣(II), have been characterized in vertebrates (7,8). They become assembled into [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers with insect cell coexpression data arguing strongly against the formation of mixed ␣(I)␣(II)␤ 2 tetramers (8). The ␤ subunits of P4Hs are identical to the enzyme and chaperone protein disulfide isomerase (PDI) (EC 5.3.4.1) (9) and are required to maintain the ␣ subunits in a catalytically active nonaggregated conformation (10, 11). The P4H is also maintained within its correct subcellular compartment by virtue of an ER retention signal at the C terminus of PDI (11). When expressed alone in a recombinant expression system, the ␣ subunits are insoluble and inactive, whereas coexpression with PDI results in the formation of an active, soluble P4H (7, 12-14). The PDIs from different organisms can often substitute for the auth...

show abstract

Section: Cloning Of B Malayi Phy-1-threementioning

confidence: 78%

Section: Fig 8 Developmental Expression Analysis Of B Malayi Phy-1mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase

Winter

Myllyharju

Page

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The wellcharacterized vertebrate type I, II, and III collagen P4Hs (C-P4Hs) reside within the lumen of the endoplasmic reticulum and are a 2 b 2 tetramers in which the catalytic a subunits have three isoforms: a(I), a(II), and a(III) (Helaakoski et al, 1989;Annunen et al, 1997;Kukkola et al, 2003;Van Den Diepstraten et al, 2003). Animal C-P4Hs have also been identified and characterized from nematodes (Veijola et al, 1994;Friedman et al, 2000;Winter and Page, 2000;Merriweather et al, 2001;Myllyharju et al, 2002;Riihimaa et al, 2002;Winter et al, 2003) and Drosophila melanogaster (Annunen et al, 1999;Abrams and Andrew, 2002). A second family of animal P4Hs has a cytoplasmic and nuclear location.…”

Section: Introductionmentioning

confidence: 99%

Chlamydomonas reinhardtiiHas Multiple Prolyl 4-Hydroxylases, One of Which Is Essential for Proper Cell Wall Assembly

et al. 2007

View full text Add to dashboard Cite

Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a soluble 29-kD monomer that effectively hydroxylated in vitro both poly(L-Pro) and synthetic peptides representing Pro-rich motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1 by RNA interference led to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7 layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their fibrous shafts. Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate specificities and functions.

show abstract

“…The PHY-1 polypeptides from both species have an 18-amino-acid C-terminal extension (Fig. 1), which is also found in the PHY-1 polypeptide of the filarial nematode Onchocerca volvulus (34), but not in that of Brugia malayi (31), nor in the Caenorhabditis PHY-2 polypeptides or vertebrate and Drosophila subunits (9-12, 15, 17, 30).…”

Section: Resultsmentioning

confidence: 91%

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

et al. 2007

View full text Add to dashboard Cite

show abstract

Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase

Cited by 17 publications

References 31 publications

A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase

A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase

Chlamydomonas reinhardtiiHas Multiple Prolyl 4-Hydroxylases, One of Which Is Essential for Proper Cell Wall Assembly

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

Contact Info

Product

Resources

About