Abstract
The collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in the X-Pro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are alpha(2)beta(2) tetramers with the catalytic activity residing in the alpha subunits. The beta 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 alpha 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.
Highlights
Biosynthesis of vertebrate collagens requires processing by up to eight specific intra- and extracellular posttranslational enzymes [1]
In this article we describe the identification of a prolyl 4-hydroxylase (P4H) phy-1 gene from B. malayi, characterize the molecular and enzymatic properties of the recombinant B. malayi P4H produced in an insect cell expression system, and examine the expression profile and putative function of the B. malayi phy-1 gene by heterologous expression in the model nematode C. elegans
The recombinant B. malayi PHY-1 assembles into an active soluble P4H tetramer when expressed in insect cells in the absence of protein disulfide isomerase (PDI)
Summary
Biosynthesis of vertebrate collagens requires processing by up to eight specific intra- and extracellular posttranslational enzymes [1]. Two ␣ subunit isoforms, ␣(I) and ␣(II), have been characterized in vertebrates [7, 8] They become assembled into [␣(I)]22 and [␣(II)]22 tetramers with insect cell coexpression data arguing strongly against the formation of mixed ␣(I)␣(II)2 tetramers [8]. Mutations in collagens forming the cuticle and in the enzymes involved in collagen biosynthesis can result in lethality and severe alterations to body shape, as illustrated by the C. elegans sqt-3 [18] and bli-4 [19] mutant phenotypes. The P4Hs in C. elegans that are involved in the synthesis of cuticle collagens are formed from the ␣ subunits PHY-1 and PHY-2 and the  subunit PDI-2 [14, 15]. Genetic disruption of phy-1 and phy-2 simultaneously, or pdi-2 singly, results in embryonic lethality in which embryos develop normally until the first cuticle is required to maintain the elongated worm
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