Introduction Prolyl hydroxylation is a widely observed post
Prolyl hydroxylation is a widely observed post translational modification in collagen, an abundant animal protein. The high content (~13% per chain of the triple helical structure) of 4-hydroxyproline (Hyp,O) in collagen, togather with the natural abundance of collagen has led to the estimation that “Hyp is more abundant in animals, than seven common amino OTX-015 residues(Cys, Gln, His, Phe, Trp, Tyr)” . Hydroxylation of proline to yield 3-hydroxy proline is a rare modification in collagen, catalysed by specific prolyl-3-hydroxylase(P3Hs) [2,3]. Hydroxylation of peptidyl proline residues is also important in cellular oxygen sensing involving hypoxia inducing factor(Hif) [4,5] and in proteins with collagen like domains . Proline hydroxylation of ribosomal proteins may also serve important regulatory functions  and has been implicated in the development of the parasitic nematode Brugia malayi . The post translational formation of 4-hydroxy proline is important in the extensins, which are glycoproteins involved in plant cell walls, where the hydroxylated residues serve as sites for O-glycosylation . Proline hydroxylation is also widely observed in peptides of microbial origin  and in conotoxins, which are the major constituent of marine cone snail venom . The enzymatic hydroxylation of peptidyl proline residues at position 4 (Cγ) is catalysed by prolyl-4-hydroxylase (P4H), a member of the non-heme iron(II)-2-oxoglutarate dependent dioxygenases . The animal P4H enzymes have been shown to be α2β2 tetramers, with the α-chain containing both the peptide substrate binding domain and the catalytic centre, while the β-subunit independently functions as a protein disulfide isomerase . A cartoon of the α2β2 tetramer based on a model derived from low angle X-ray scattering  and available crystal structures of independent domains [15,16] is shown in Fig. 1. A crystal structure of the intact α2β2 tetramer has not been reported. Plant P4H enzymes are significantly smaller in size and are active as monomers [16,17]. A homodimeric P4H, which modifies elongation factor Tu has been characterised from Bacillus anthracis [18,19]. Proline hydroxylation is one of the most common post translational modifications in the peptides derived from marine cone snail venom [11,20]. Conotoxins are a structurally diverse class of peptides, targeting a wide range of membrane receptors and ion channels resulting in rapid paralysis of prey [, , , ]. While, disulfide isomerases have been identified experimentally from venom  and a large number of sequences determined by transcriptomic analysis [25,26], there are, to the best of our knowledge no reports on the sequences of cone snail P4H enzymes. The identification of a protein on 2D gel electrophoresis of a C. geographus venom duct extract, as P4H has been reported in a proteomic analysis . We report in this communication sequences of prolyl-4-hydroxylase obtained by mining transcriptomic data from seven species C. amadis, C. monile, C. araneosus, C. frigidus, C. ebraeus, C. miles, and C. litteratus. Functional annotation of the Conus P4H sequences has been established by comparing the conservation of key residues involved in the structure and function of mammalian prolyl-4-hydroxylase. We also present the characterisation of conotoxins containing multiple proline residues from C. amadis, providing examples of extensive hydroxylation, site specific modification, and the absence of conversion to hydroxy proline, suggesting that conotoxin precursor protein sequences may determine the extent and site specificity of post translational proline hydroxylation.
Material and methods
Discussion The high degree of conservation of the prolyl-4-hydroxylase active site between the cone snail sequences and the Homo sapiens, Gallus gallus and Bos taurus prolyl-4-hydroxylase, suggests that only the hydroxylation prone proline residue is in direct contact with the active site, thereby permitting a wide range of proline containing substrates to be post translationally modified. The lack of any major difference between the Conus P4H active site residues and those of vertebrate enzymes suggests substrate binding and proline positioning may not be responsible for the observed region- selectivity of hydroxylation. Local backbone conformations at the site of hydroxylation may be important, with the both extended conformations and beta turns  having been implicated for collagenous peptide substrates.