Peter Bruckner

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

Abstract Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.

Supramolecular Interactions in the Dermo-epidermal Junction Zone: ANCHORING FIBRIL-COLLAGEN VII TIGHTLY BINDS TO BANDED COLLAGEN FIBRILS*

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.

The epidermal basement membrane is a composite of separate laminin- or collagen IV-containing networks connected by aggregated perlecan, but not by nidogens

Background: The supramolecular assemblies of basement membranes are incompletely understood. Results: Basement membranes are suprastructural composites of two separable, alloyed networks containing collagens IV or laminins. Conclusion: The laminin- and collagen IV-containing networks are “spot-welded” by perlecan aggregates, but not by nidogens. Significance: Biological properties of supramolecular assemblies, such as basement membranes, go beyond the sum of individual macromolecular attributes. The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.

Collagen XII Interacts with Avian Tenascin-X through Its NC3 Domain

Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding.

Disorder of collagen metabolism in a patient with osteogenesis imperfecta (lethal type): increased degree of hydroxylation of lysine in collagen types I and III

Abstract. Types I, II and III collagen were isolated from calvarium, skin and cartilage from a patient with recessive lethal osteogenesis imperfecta. The distribution of the various collagen types was normal in all three tissues. The α‐chains were purified by molecular sieve and ion‐exchange chromatography and were found to differ from the corresponding α‐chains of age‐matched controls only in that the α1(I), α2 and α1 (III) chains contained higher amounts of hydroxylysine with proportionally less lysine. α1(II) was normal. The excess hydroxylysine residues were all glycosylated in the case of α1(I) chains, but only partly so for the α2 chains. Similar observations were made with collagen from fetuses at various stages of development. In these fetuses, however, the increase in the degree of hydroxylation of lysine in αl(I), α2 and αl(III) varied with age, being highest in the youngest fetus. Seen in the context of embryonic development, the collagen of the patient would correspond to that of a fetus younger than 18 weeks, and one could speculate that the defect seen in this patient is the result of a disturbed process of maturation of connective tissue.

Macromolecular Specificity of Collagen Fibrillogenesis: Fibrils of Collagens I and XI Contain a Heterotypic Alloyed Core and a Collagen I-Sheath

Suprastructures of the extracellular matrix, such as banded collagen fibrils, microfibrils, filaments, or networks, are composites comprising more than one type of macromolecule. The suprastructural diversity reflects tissue-specific requirements and is achieved by formation of macromolecular composites that often share their main molecular components alloyed with minor components. Both, the mechanisms of formation and the final macromolecular organizations depend on the identity of the components and their quantitative contribution. Collagen I is the predominant matrix constituent in many tissues and aggregates with other collagens and/or fibril-associated macromolecules into distinct types of banded fibrils. Here, we studied co-assembly of collagens I and XI, which co-exist in fibrils of several normal and pathologically altered tissues, including fibrous cartilage and bone, or osteoarthritic joints. Immediately upon initiation of fibrillogenesis, the proteins co-assembled into alloy-like stubby aggregates that represented efficient nucleation sites for the formation of composite fibrils. Propagation of fibrillogenesis occurred by exclusive accretion of collagen I to yield composite fibrils of highly variable diameters. Therefore, collagen I/XI fibrils strikingly differed from the homogeneous fibrillar alloy generated by collagens II and XI, although the constituent polypeptides of collagens I and II are highly homologous. Thus, the mode of aggregation of collagens into vastly diverse fibrillar composites is finely tuned by subtle differences in molecular structures through formation of macromolecular alloys.

Isolation of unhydroxylated type I procollagen folding of the protein in vitro

Abstract Tendons from 17-day-old chick embryos were incubated with α,α′-dipyridyl so that they synthesized type I protocollagen, the unhydroxylated form of type I procollagen. The incubation medium was cooled to 4 to 15 °C so that the protocollagen secreted into the medium folded into a triple-helical conformation, and the protein was purified by ion-exchange chromatography and velocity sedimentation. The isolated protocollagen had the expected amino acid composition in that it was similar to procollagen but it contained only 1 to 8 residues per 1000 of hydroxyproline and 1 residue per 1000 of hydroxylysine. At 15 °C, the circular dichroism spectrum of the protein was similar to that of triple-helical procollagen or collagen, and the protein underwent a thermal transition with a midpoint of about 25.5 °C. The folding and unfolding of the protein was reversible in that on recooling the denatured protein, essentially all the initial helicity was recovered. The folded form of the protein, however, did not consist of perfectly aligned triple-helical structures. Analysis of the circular dichroism spectrum demonstrated that the specific mean residue ellipticities of the peaks at 221 and 198 nm were less than the expected values of native procollagen. Also, the thermal transition for protocollagen was broader than that observed with procollagen. When examined by zonal centrifugation at 4 °C, most of the protein sedimented with the same apparent sedimentation coefficient as native procollagen but the peak of protocollagen was asymmetric and tailed toward the bottom of the gradient. When incubated with prolyl hydroxylase, some of the prolyl residues were hydroxylated. When incubated with a mixture of trypsin and chymotrypsin, about 40% of the protein was cleaved under conditions in which triple-helical procollagen and collagen resisted digestion. The results demonstrated that although type I protocollagen folds and refolds reversibly in vitro, about half of the molecules present in the folded form of the protein contain short regions which are not in a triple-helical conformation.

Binde- und Stützgewebe

Das Bindegewebe durchzieht den gesamten Organismus. Um den vielfaltigen Aufgaben als Gerust- und Stutzsubstanz gerecht zu werden kommt es in den unterschiedlichsten Auspragungen vor. Dazu gehoren feste Strukturen wie Knorpel, Sehnen und Bander, aber auch das aus locker gepackten fibrillaren Strukturen bestehende interstitielle Bindegewebe, das den Extrazellularraum ausfullt und Organe umgibt. Das Bindegewebe ist aber weit mehr als nur das strukturgebende Element des Korpers. Eine Vielzahl von extrazellularen Matrix-Molekulen binden uber spezifische Rezeptoren an Zellen und beeinflussen Wachstum, Differenzierung und Funktion fast aller Zellen des Korpers. Eindrucksvoll konnte dies an Mausen gezeigt werden, bei denen Rezeptoren oder deren extrazellulare Liganden inaktiviert waren. Im Extremfall kam es nicht einmal zur Ausbildung des zweiblattrigen Keimblatts.

Aggregation of a type I collagen precursor containing N-terminal propeptides.

Aggregation properties of type I pNcollagen from dermatosparactic sheep skin were studied by electron microscopy and turbidity measurements. pNcollagen was less soluble than collagen at low temperatures. Upon heating to 37 degrees C, it polymerized into thin, flat fibrils showing the same periodicity as collagen but with a less distinct gap-overlap pattern. The apparent critical concentration was 0.01-0.03 mg/ml at temperatures between 32-37 degrees C and was higher than that for collagen under similar conditions. The total turbidity change with heat polymerization of the pNcollagen was less than with heat polymerization of collagen under the same conditions. Also, the total change in turbidity showed a different temperature dependence and the kinetics of the reaction were slower than with collagen. However, the effects of protein concentration, ionic strength and pH on the rate of polymerization of pNcollagen were similar to those observed with collagen. The kinetics of polymerization of pNcollagen were explained by a model based on nucleation and growth processes which permits end-to-end association of fibrils, but only limited lateral association because of the presence of the N-terminal propeptides.

Lateral Growth Limitation of Corneal Fibrils and Their Lamellar Stacking Depend on Covalent Collagen Cross-linking by Transglutaminase-2 and Lysyl Oxidases, Respectively

Background: Mechanisms of growth limitation and lamellar stacking of collagen fibrils in cornea remain elusive. Results: Covalent collagen cross-links are formed by catalysis involving both lysyl oxidases and tissue transglutaminase-2. Conclusion: Aldehyde-derived and isopeptide cross-linking of collagen determine lamellar stacking and lateral fibril growth, respectively. Significance: Two types of covalent collagen cross-linking are indispensable for correct corneal morphogenesis. Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ∼35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea.