The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.     

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule’s size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.     

Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties.

Structural stability of the extracellular matrix is primarily a consequence of fibrillar collagen and the extent of cross-linking. The relationship between collagen self-assembly, consequent fibrillar shape and mechanical properties remains unclear. Our laboratory developed a model system for the preparation of self-assembled type I collagen fibers with fibrillar substructure mimicking the hierarchical structures of tendon. The present study evaluates the effects of pH and temperature during self-assembly on fibrillar structure, and relates the structural effects of these treatments on the uniaxial tensile mechanical properties of self-assembled collagen fibers. Results of the analysis of fibril diameter distributions and mechanical properties of the fibers formed under the different incubation conditions indicate that fibril diameters grow via the lateral fusion of discrete approximately 4 nm subunits, and that fibril diameter correlates positively with the low strain modulus. Fibril diameter did not correlate with either the ultimate tensile strength or the high strain elastic modulus, which suggests that lateral aggregation and consequently fibril diameter influences mechanical properties during small strain mechanical deformation. We hypothesize that self-assembly is mediated by the formation of fibrillar subunits that laterally and linearly fuse resulting in fibrillar growth. Lateral fusion appears important in generating resistance to deformation at low strain, while linear fusion leading to longer fibrils appears important in the ultimate mechanical properties at high strain.     

Amyloid fibril formation from full-length and fragments of amylin

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.     

Monomer and oligomer of type I collagen: molecular properties and fibril assembly

Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

Cultured epidermis influences the fibril organization of purified type I collagen gels

Purified type I collagen gel used as culture substrate was composed of unstriated fibrils. Before culture, gel fragments were coated with culture medium with or without fetal calf serum (FCS+ coated or FCS- coated gels). Each gel fragment was apposed to a fragment of frog skin at the medium/air interface in Trowell culture chamber. After 7 days at 20 degrees C, the coated gels were covered with newly formed epidermis containing fibronectin localized around the keratinocytes, whose morphology was considerably modified. Fibroblast-shaped keratinocytes were localized in the anterior zone of the newly formed epidermis on FCS+ gels. The long axis of the cells was parallel to the gel surface, where numerous unstriated fibrils were located. Polyhedral keratinocytes were located in the posterior zone on FCS+ gels or the anterior and posterior zones on FCS- gels with the long axis perpendicular to the gel surface. Numerous cross-striated fibrils were found under the cultured keratinocytes in the vicinity of the basal filipodia. This model is useful for the study of collagen gel reorganization by keratinocytes.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

The interaction of decorin core protein fragments with type I collagen.

To further define the molecular interaction between decorin and type I collagen we generated a 20 kD fragment containing the N-terminal half of the core protein by Endoproteinase Arg C digestion and a 40 kD fragment including all leucine-rich repeats in the central part of decorin core by cleavage with 2-nitro-5-thiocyanobenzoate. The fragments did not influence collagen fibril formation, even at high concentration, and radioactive fragments showed little binding to collagen fibrils. Our observations suggest that neither the N-terminal half nor the central leucine-rich repeats of the decorin core protein can, by itself, interact fully with fibrillar collagen.     

Binding of soluble type I collagen molecules to the fibroblast plasma membrane.

Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.     

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.     

Distinct modes of collagen type I proteolysis by matrix metalloproteinase (MMP) 2 and membrane type I MMP during the migration of a tip endothelial cell: insights from a computational model

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extracellular matrix (ECM) structural proteins under physiological and pathological conditions. During sprouting angiogenesis, the MMPs expressed by a single "tip" endothelial cell exhibit proteolytic activity that allows the cells of the sprouting vessel bud to migrate into the ECM. Membrane type I matrix metalloproteinase (MT1-MMP) and the diffusible matrix metalloproteinase MMP2, in the presence of the tissue inhibitor of metalloproteinases TIMP2, constitute a system of proteins that play an important role during the proteolysis of collagen type I matrices. Here, we have formulated a computational model to investigate the proteolytic potential of such a tip endothelial cell. The cell expresses MMP2 in its proenzyme form, pro-MMP2, as well as MT1-MMP and TIMP2. The interactions of the proteins are described by a biochemically detailed reaction network. Assuming that the rate-limiting step of the migration is the ability of the tip cell to carry out proteolysis, we have estimated cell velocities for matrices of different collagen content. The estimated velocities of a few microns per hour are in agreement with experimental data. At high collagen content, proteolysis was carried out primarily by MT1-MMP and localized to the cell leading edge, whereas at lower concentrations, MT1-MMP and MMP2 were found to act in parallel, causing proteolysis in the vicinity of the leading edge. TIMP2 is a regulator of the proteolysis localization because it can shift the activity of MT1-MMP from its enzymatic toward its activatory mode, suggesting a tight mechanosensitive regulation of the enzymes and inhibitor expression. The model described here provides a foundation for quantitative studies of angiogenesis in extracellular matrices of different compositions, both in vitro and in vivo. It also identifies critical parameters whose values are not presently available and which should be determined in future experiments.     

Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 "null" allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5' donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.