Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor.

The nematode Caenorhabditis elegans represents an excellent model in which to examine nematode gene expression and function. A completed genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of nematode genes. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. Many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterized bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed

Role of prolyl hydroxylation in the molecular interactions of collagens

Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour. © 2019 The Author(s).</p

Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris

It was recently reported that co-expression of the proal(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J, 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only similar to 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human pro alpha 1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical pro alpha 1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments, The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved

Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris

It was recently reported that co-expression of the proal(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J, 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only similar to 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human pro alpha 1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical pro alpha 1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments, The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved.</p

Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are \u3b1(2)\u3b2(2) tetramers with three isoforms of the catalytic \u3b1 subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1\u3b1, HIF-2\u3b1, or the von Hippel-Lindau protein. The data obtained showed that C-P4H \u3b1(I) and \u3b1(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change

Gene expression and matrix turnover in overused and damaged tendons

Chronic, painful conditions affecting tendons, frequently known as tendinopathy, are very common types of sporting injury. The tendon extracellular matrix is substantially altered in tendinopathy, and these changes are thought to precede and underlie the clinical condition. The tendon cell response to repeated minor injuries or “overuse” is thought to be a major factor in the development of tendinopathy. Changes in matrix turnover may also be effected by the cellular response to physical load, altering the balance of matrix turnover and changing the structure and composition of the tendon. Matrix turnover is relatively high in tendons exposed to high mechanical demands, such as the supraspinatus and Achilles, and this is thought to represent either a repair or tissue maintenance function. Metalloproteinases are a large family of enzymes capable of degrading all of the tendon matrix components, and these are thought to play a major role in the degradation of matrix during development, adaptation and repair. It is proposed that some metalloproteinase enzymes are required for the health of the tendon, and others may be damaging, leading to degeneration of the tissue. Further research is required to investigate how these enzyme activities are regulated in tendon and altered in tendinopathy. A profile of all the metalloproteinases expressed and active in healthy and degenerate tendon is required and may lead to the development of new drug therapies for these common and debilitating sports injuries

Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein

Critical Early Roles for col27a1a and col27a1b in Zebrafish Notochord Morphogenesis, Vertebral Mineralization and Post-embryonic Axial Growth

Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development.We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically.Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype

Insights on the Evolution of Prolyl 3-Hydroxylation Sites from Comparative Analysis of Chicken and Xenopus Fibrillar Collagens

Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except α1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only α2(I) and α2(V) chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues