Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 113-123).

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils.

La maturation des collagènes fibrillaires est régulée par la libération protéolytique des propeptides qui conduit à la formation spontanée de fibres, à partir des molécules de collagène mature. Les fibres de collagène ainsi formées confèrent résistance et solidité aux tissus. Les métalloprotéases Tolloïdes sont les principales enzymes responsables de la maturation C-terminale des procollagènes. Ces protéases possèdent de nombreux autres substrats et se distinguent par un mode de régulation original, l’utilisation de régulateurs « substrats-spécifiques », qui leur permet de moduler leur activité vis-à-vis de ces différents substrats. Parmi ces régulateurs, les Procollagen C-Proteinase Enhancers (PCPE-1 et 2) semblent jouer un rôle important car ils augmentent l’activité de clivage du C-propeptide des procollagènes jusqu’à 20 fois. Même si PCPE-1 a été découvert en 1985, son mode d’action n’est toujours pas compris. Le principal objectif de cette thèse était donc de mieux comprendre le mécanisme de l’activation. Nous avons commencé par identifier la région minimale de PCPE-1 capable d’activer les Tolloïdes et démontré une très forte coopérativité entre les domaines de cette région. Par ailleurs, nous avons mis en évidence, pour la première fois, un nouveau rôle physiologique pour le domaine C-terminal de PCPE-1 (NTR). Concernant les autres partenaires du complexe de maturation, nous avons observé une interaction d’affinité modérée entre PCPE-1 et la protéase Tolloïde appelée BMP-1 et montré que PCPE-1 se fixe uniquement sur la partie C-terminale du procollagène. Enfin, nous avons mis en évidence que d’autres métalloprotéases, les Méprines, pourraient également jouer un rôle dans la maturation de collagènes fibrillaires en étant régulées négativement par les PCPEs. L’ensemble de ces résultats nous a permis de proposer un nouveau schéma d’interaction pour les PCPEs et de faire de nouvelles hypothèses concernant leur mécanisme d’action / The maturation of fibrillar collagens is a tightly regulated process controlled by two proteolytic cleavages that remove the propeptide regions from procollagen precursors leading to spontaneous assembly of mature collagen molecules into fibrils. These fibrils provide tensile strength and toughness to connective tissues and consequently to the organism itself. The group of extracellular metalloproteinases mostly responsible for procollagen processing are the tolloids. As these enzymes have several functions other than procollagen processing, their activities on different substrates are controlled by a growing number of substrate-specific regulators. The most prominent of these regulators are the procollagen C-proteinase enhancers (PCPEs), of which PCPE-1 is a 55 kDa glycoprotein composed of two CUB and a C-terminal NTR domain, which is capable of enhancing the proteolytic activity of tolloids up to 20-fold. Even though PCPE-1 has been known since 1985 the molecular mechanism of enhancement is still unclear. The aim of this thesis was to understand and characterize this mechanism with the aid of biochemical and biophysical methods. We have identified the minimal unit responsible for enhancing activity. In addition, we propose a mechanism for how the subdomains responsible for enhancement cooperatively bind to procollagen substrates. Furthermore, we have for the first time been able to identify a possible physiological function of the NTR domain. Also, we have identified meprins as new players involved in procollagen processing and this has given valuable insights in the mechanism of action of PCPEs. Finally, we have been able to demonstrate that the interaction of PCPE-1 with procollagen is mostly limited to the C-propeptide region. Based on these findings we propose a new hypothetical interaction mechanism for PCPE-1

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [104]-114).

In an attempt to elucidate the transcriptional mechanisms that regulate the expression of the human α2(I) procollagen gene, cis-acting DNA-elements within the proximal promoter were identified and their corresponding trans-acting factors characterised. The fibroblast cell lines used in this study had previously been transformed with either simian virus 40 (SVWI-38) or by γ-radiation (CT-1). The SVWI-38 fibroblasts do not produce any α2(I) collagen chains, whereas the CT-1 cell line produces normal type I collagen. Previous studies suggested that trans-acting factor(s) may be responsible for the inactivation of the α2(I) procollagen gene in SVWI-38 fibroblasts (Parker et. al. (1989) J. Biol. Chem 264, 7147-7152; Parker et. al. (1992) Nucleic Acids Res. 20, 5825-5830). In this study, the SVWI-38 proximal promoter (-350 to +54) was sequenced and shown to be normal, thereby ruling out any possibility that mutations within this region was responsible for inactivation of the gene.

La balance entre synthèse et dégradation des collagènes joue un rôle crucial dans le développement de pathologies de cicatrisation. Les collagènes fibrillaires sont synthétisés et sécrétés sous forme de précurseurs dans l'espace extracellulaire. Ils subissent alors une maturation protéolytique par des métalloprotéases, telles que les « BMP-1/Tolloid-like » (BTPs), permettant la formation de fibres de collagène. Ce clivage protéolytique est stimulé de façon spécifique par deux glycoprotéine, les « Procollagen C-Proteinase Enhancers » (PCPE-1 et -2). Ainsi, l'objectif de mon travail de recherche a été d'étudier les rôles de PCPE-1 et -2 dans la cicatrisation cutanée. De façon intéressante, nous avons montré que les BTPs et PCPE-1 étaient exprimés au niveau du derme et que leur expression augmentait pendant la première semaine de cicatrisation. PCPE-1 est principalement exprimée par les fibroblastes alors que PCPE-2 est retrouvée dans les kératinocytes. De plus, nous avons montré que PCPE-2 était abondante dans les cellules myéloides, suggérant un rôle pendant la phase inflammatoire de cicatrisation. Nos résultats montrent que PCPE-2 ne stimule pas efficacement la maturation des procollagènes fibrillaires et inhibe même l'activité de BMP-1 sur des substrats non-collagéniques, suggérant un rôle inattendu de PCPE-2. Enfin, aucune différence majeure n'a été observée dans la peau de souris déficientes en PCPE-2 au cours de la cicatrisation, suggérant que PCPE-1 serait le principal stimulateur impliqué dans la maturation et le dépôt des fibres de collagènes. Ainsi, ce travail suggère que PCPE-1 et -2 joueraient des rôles distincts dans la peau et pendant la cicatrisation cutanée / The imbalance between collagen assembly and degradation during skin wound healing is a major factor contributing to wound healing pathologies. Fibrillar collagen precursors are synthesized by fibroblasts and processed in the extracellular space by specific metalloproteases (e.g. BMP-1/Tolloid-like Proteinases, or BTPs) to form collagen fibrils. These proteolytic maturations can be efficiently stimulated by two glycoproteins, Procollagen C-Proteinase Enhancers (PCPE-1 and -2). The main goal of our study was to analyze the possible redundant and specific roles of the two PCPE proteins during wound healing.Interestingly, both BTPs and PCPE-1 were found to be expressed in the dermis and to be significantly increased during the first week after injury. Surprisingly, PCPE-1 is mainly expressed by dermal fibroblasts, whereas PCPE-2 is lowly expressed in fibroblasts and more abundant in basal keratinocytes. Moreover, PCPE-2 appears to be expressed by myeloid cells suggesting that PCPE-2 might rather play a role during the inflammatory phase of wound healing. In addition, our results indicate that recombinant PCPE 2 does not efficiently enhance their proteolytic maturation of fibrillar procollagens and can even inhibit the action of BMP-1 on other non-collagenous substrates, suggesting a differential and unexpected role of PCPE-2. Finally, the in vivo role of PCPE-2 was investigated using a Pcolce2 knockout model; skin morphology and wound healing were not affected by PCPE-2 loss, indicating that PCPE-1 is the main enhancer involved in collagen deposition during wound healing. Thus, this work suggests that PCPE-1 and -2 play distinct roles in the skin and during wound healing

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 133-147). Also available on the Internet.

Abstract Malignant tissue growth induces marked biochemical and structural changes in the extracellular matrix of the tumour and its surrounding tissues. In the present study, we evaluated the prognostic value of the serum concentration of the markers of synthesis of type I collagen (PICP, PINP) and type III collagen (PIIINP) as well as the marker of type I collagen degradation (ICTP) and compared them with the conventional indicators of prognosis (clinical stage, grade of differentiation, histological subtype, residual tumour load and the age of the patient). The prognostic value of peritoneal cytological findings at operation was an additional object in our studies. High preoperative serum ICTP (>5.6μg/L) and PIIINP (>3.2μg/L) concentrations and a low PICP:PINP ratio (>2) correlated with poor prognosis in ovarian carcinoma in univariate analysis and in multivariate analysis when each variable was analyzed separately with the conventional factors. However, ICTP concentration was the only prognostic variable in multivariate analysis including PIIINP, PINP, ICTP and CA125. When analyzed with conventional prognostic factors (clinical stage, grade, residual tumour, presence of ascites, histology), clinical stage and ICTP were independent indicators of prognosis. In addition, malignant cells in the peritoneal fluid aspirate at primary operation, grade and the age of the patient predicted poor prognosis in multivariate analysis. Postoperative serum ICTP concentration 9-months after the operation was the strongest prognostic factor as compared to the preoperative ICTP and CA125 values and clinical variables. These results indicate that serum collagen metabolites, especially ICTP, are indicators of prognosis in epithelial ovarian cancer. The present ICTP-test does not detect the degradation products of immature type I collagen, the dominating form in ovarian cancer tissue. Therefore, the excess ICTP in invasive ovarian cancer might originate through the degradation of trivalently matured collagens in non-malignant tissues surrounding the malignancy. ICTP may thus be an indicator of invasive properties of the tumor and its determination opens up new perspective to predict the clinical outcome of ovarian cancer.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.