Molecular structure and physical properties of type IV collagen in solution

The physical properties of intact type IV collagen from the mouse EHS sarcoma were studied in acid solution using laser light scattering and viscometry. The experimentally observed values of molecular weight, translational diffusion coefficient, particle scattering factor at 175.5° and a wavelength of 633 nm and intrinsic viscosity at 22°C were 532000, 0.66 × 10⁻⁷cm²s⁻¹, 0.492 and 74.7 ml/g respectively. Plots of $\text{Kc/}\text{R}{}_0$ versus collagen concentration were linear with a slope of approximately 0, indicating that under the conditions studied, type IV collagen molecules do not form supra-molecular aggregates. Experimentally determined translational diffusion coefficients closely approximated the calculated value for a rod-like molecule 424 nm long and 1.5 nm in diameter. Based on this observation, it is concluded that the type IV collagen molecule translates like a bent rigid rod similar to the interstitial collagens. However, the low intrinsic viscosity and larger value of the particle scattering factor for type IV collagen molecules in comparison with the interstitial collagens indicate that type IV collagen is considerably more flexible. Physical measurements on molecules in solution are consistent with a model of the type IV molecule containing numerous flexible bends with bend angles less than 125°. It is concluded that the type IV collagen molecule behaves like a worm-like rod in solution.     

Kinetics of hydrolysis of type I,II, and III collagens by the class I and IIClostridium histolyticum collagenases

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Collagen crosslinks: isolation of reduced N -hexosylhydroxylysine from borohydride-reduced calf skin insoluble collagen

Treatment of calf skin insoluble collagen with NaB³H₄ followed by acid hydrolysis and ion-exchange chromatography yields a new compound which is prominent in the chromatograms of several collagens. We now describe this compound as reduced N^?-hexosylhydroxylysine. It appears to arise by reduction of the Schiff base between the carbonyl moiety of a hexose and the ?-amino group of hydroxylysine; the postulated structure was derived from both high- and low-resolution mass spectrometry and by comparison with synthetic N^?-galactosylhydroxylysine. Conceivably, the unreduced compound may be a crosslink, uniting collagen and glycoproteins or proteoglycans in the connective tissue.     

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.     

Cross-Linking and flourescence of pyrene-labeled collagen

The pyrene-labeling of acid-soluble (type I) and acid-insoluble collagens from young and old rat tail tendon has been investigated. The pyrene excimer fluorescence is associated with stabilized pyrene labels bound to two adjacent aldehydes in monomeric young collagens. Polymeric young collagens, as well as monomeric and polymeric old collagens, tend to lose this specific arrangement. This is shown by salt and new chromatographic fractionation of monomeric and polymeric collagens. During denaturation, pyrene labels are released from saturated aldehydes in both α₁ and α₂ chains. This unstable pyrene-labeling is stabilized by NaBH₄ reduction of the hydrazone bonds between aldehyde groups and pyrene-containing hydrazines. This stabilization reveals that α₁ contains more aldehyde groups than does α₂ in young collagen. Pepsin-solubilized, acid-insoluble collagens are partly cross-linked and, like acid-soluble collagens, exhibit the fluorescence of pyrene aggregates probably located atunidentified cross-links, different from unsaturated aldehyde-containing cross-links in acid-soluble collagens.     

Analysis of an ascidian integrin provides new insight into early evolution of collagen recognition

αI domain integrins have been found in the ascidian Ciona intestinalis. We produced Ciona α1I domain as a recombinant protein. It did not recognize fibril-forming collagens or bind to GFOGER or other similar motifs in triple-helical peptides. No GFOGER motifs were found in Ciona collagens. As Ciona α1I bound to collagen IX, we propose that before the emergence of GFOGER-dependent collagen receptors in vertebrates, αI domain integrins might have been able to bind to collagen with alternative mechanisms.     

Association of collagen with preimplantation and peri-implantation mouse embryos

By immunofluorescence analyses, we have determined that Type III procollagen, Type III collagen, and B and C chains of basement membrane collagen are associated with preimplantation mouse embryos. Type III collagen and procollagen appear to be associated with embryos at the 4-cell stage and beyond, whereas antibodies to B and C collagen chains bind to 2-cell and later embryos. All of these collagen types are detected in increasing amounts as embryos develop in a defined medium, indicating that the embryo is capable of their synthesis. By the blastocyst stage, the collagens are primarily localized intercellularly. Cells of the inner cell mass (ICM) also bind collagen antibodies. When isolated ICMs become two-layered, both the inner presumptive ectoderm layer and the outer primitive endoderm layer react with antibodies to Type III collagen and procollagen. The endoderm cells also react avidly with antibodies to B- and C-chain collagens. Preimplantation embryos and ICMs fail to react with antibodies to Types I and II collagen. During peri-implantation stages, blastocysts continue to react with antibodies to Type III and basement membrane collagens. There is no obvious relationship between the intensity of immunofluorescence and the change in the blastocyst surface from nonadhesive to adhesive. Furthermore, blastocysts prevented from undergoing implantation-related events in utero and in vitro react extensively with collagen antibodies. Blastocyst surface collagens might, nevertheless, play a role in implantation by undergoing organizational changes.     

Corneal and scleral type I collagens: analyses of physical properties and molecular flexibility

The physical properties of type I collagen were studied by electron microscopy of rotary shadowed collagen molecules and laser light scattering techniques. The physical properties, molecular structure and flexibility of type I collagen molecules from two structurally and functionally different connective tissues, cornea and sclera, were similar when measured in HCl, pH 2.0. The molecular weights were 328 and 298 × 10² for corneal and scleral type I collagen, respectively, while the values of T_M were 33.7°C for both preparations. These values were in agreement with those obtained for other type I collagens. The higher level of glycosylation in corneal versus scleral type I collagen did not significantly modify the physical properties of type I collagen in acid solution or the charge distribution along the molecule as determined from the positively stained SLS banding patterns. Our morphological studies indicated that the collagen molecule, although relatively flexible based on electron microscopy, behaved as a long thin rod in solution. The mean end-to-end distances measured from electron micrographs were 253 and 256 nm for corneal and cler type I collagen, respectively, while the molecular contour lengths were 298 and 305 nm. The translational diffusion coefficients (0.849 and 0.857 × 10^?7cm²s^?1) were consistent with the contour lengths while the reported values in the literature for the rotational diffusion coefficient of type I collagen were consistent with the end-to-end distances. The intermediate value for molecular length obtained from the particle scattering factor (277 nm) reflects contributions from all possible molecular configurations.     

Molecular composition and function of integrin-based collagen glues—Introducing COLINBRIs

Background

Despite detailed knowledge about the structure and signaling properties of individual collagen receptors, much remains to be learned about how these receptors participate in linking cells to fibrillar collagen matrices in tissues. In addition to collagen-binding integrins, a group of proteins with affinity both for fibrillar collagens and integrins link these two protein families together. We have introduced the name COLINBRI (COLlagen INtegrin BRIdging) for this set of molecules. Whereas collagens are the major building blocks in tissues and defects in these structural proteins have severe consequences for tissue integrity, the mild phenotypes of the integrin type of collagen receptors have raised questions about their importance in tissue biology and pathology.

Scope of review

We will discuss the two types of cell linkages to fibrillar collagen (direct- versus indirect COLINBRI-mediated) and discuss how the parallel existence of direct and indirect linkages to collagens may ensure tissue integrity.

Major conclusions

The observed mild phenotypes of mice deficient in collagen-binding integrins and the relatively restricted availability of integrin-binding sequences in mature fibrillar collagen matrices support the existence of indirect collagen-binding mechanisms in parallel with direct collagen binding in vivo.

General significance

A continued focus on understanding the molecular details of cell adhesion mechanisms to collagens will be important and will benefit our understanding of diseases like tissue- and tumor fibrosis where collagen dynamics are disturbed. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Exposure to Mimivirus Collagen Promotes Arthritis

Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

Temporal and spatial transitions in collagen types during embryonic chick limb development

To determine whether transitions occur in the types of collagen synthesized during embryonic chick limb development, the α chain composition of the collagens produced by whole limbs and various anatomical regions of limbs was analyzed at different stages (23–24 to 40). The tissues were incubated in the presence of ³H-proline and ³H-lysine and the α chain distribution of the purified, labeled collagens was determined by chromatography on carboxymethyl cellulose columns. We found that the stage 23–24 leg mesenchyme is producing predominantly, if not solely, an (α1)₂α2 type collagen (chain type as yet undetermined). At about stage 25–26 the limb core begins synthesizing detectable amounts of (α1)₃ collagen, which we presume to be cartilage type collagen, [α1 (II)]₃, while the outer portion of the limb largely continues to produce (α1)₂α2. The production of (α1)₃ collagen in the core progressively increases until, by stage 33 it is the only species detectable in the tibial diaphysis. Shortly thereafter (by stage 35⁺–36) (α1)₂α2 type collagen reappears in the tibial diaphysis signifying the production of bone collagen, [α1 (I)]₂α2. During the next several days of incubation, the relative proportion of (α1)₂α2 increases in the bony diaphysis while (α1)₃ remains the predominant species synthesized in the cartilaginous epiphysis.     

Hysteretic kinetic behavior of beef liver pyruvate carboxylase.

Natural abundance ¹³C nuclear magnetic resonance (nmr) spectra have been obtained for samples of a variety of native collagens by use of cross-polarization (CP) techniques which permit high resolution natural abundance ¹³C nmr spectra of solids to be obtained with high sensitivity. The CP ¹³C nmr spectra of lyophilized skin and tendon collagens consisted of two broad resonance envelopes spanning a five kHz range. Hydrated tendon collagen gave rise to a CP spectrum very similar to that obtained for the lyophilized sample, indicating that it retains its solid-like properties. In contrast, hydrated skin collagen became denatured under the conditions of the CP experiment and subsequently gave rise to a conventional high-resolution Fourier transform (FT) nmr spectrum. The CP ¹³C nmr spectrum of ivory was similar to those of lyophilized skin and tendon collagens, demonstrating the solid-like character of the collagen in dentine, whereas the CP spectrum of bovine nasal cartilage reflected the presence of highly mobile proteoglycan components in addition to relatively rigid collagen molecules. In the case of ivory, the resolution of the CP spectrum was enhanced by “magic angle” spinning to a degree approaching that of conventional FT ¹³C nmr spectra of denatured collagen in solution. Because of its ability to probe the dynamic properties of solid-like biological molecules, CP ¹³C nmr spectroscopy should be a valuable investigative tool for future studies.     

In Vitro Fabrication and Physicochemical Properties of a Hybrid Fibril from Xenogeneic Collagens

A Hybrid collagen fibril (HCF) assembled from xenogeneic collagens is a special kind of collagen fibrils in vivo and plays an important role in living systems. Inspired by nature, can a HCF form in vitro? Herein, we fabricated a new HCF by neutralizing a mixture of type I bullfrog (Rana catesbeiana Shaw) skin collagen and porcine (Sus scrofa domesticus) skin collagen with a phosphate buffer, and investigated its physicochemical properties. Self-assembly kinetics and fluorescence-quenching experiments showed that a significant intermolecular interaction and co-assembly behavior occurred between bullfrog skin collagen and porcine skin collagen, thus confirming that xenogeneic collagens can self-assemble to form HCF. Differential scanning calorimetry revealed that the thermal stability of HCF was completely different from that of the syngeneic bullfrog skin and porcine skin collagen fibrils. This finding indicated that a new kind of collagen fibril was fabricated successfully. Scanning electron microscopy and transmission electron microscopy tests showed that the diameters and D-periodicity lengths of HCF were smaller than those of the syngeneic collagen fibrils, suggesting that the morphological features of HCF were distinguished from those of the syngeneic fibril samples. Moreover, viscoelasticity of a collagen gel also changed after the self-assembly of xenogeneic collagens. Meanwhile, the obtained hybrid gel still exhibited good biocompatibility and cell proliferation properties. Finding from this work provides a new idea for the improvement or regulation of collagen-based products performance.     

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.     

Selective decrease of type I collagen synthesis in Fraser mice skin

Quantification and biosynthesis of type I and type III collagens were determined in skin of control and Fraser mice (Cat^Fraser mutation), which exhibit a genetically determined cataract. Skin organ cultures were labelled with [³H]proline. Pepsin-solubilized collagens were studied using three different approaches: (a) differential salt precipitation at neutral pH, followed by SDS-polyacrylamide gel electrophoresis; (b) differential salt precipitation at acid pH followed by SDS-polyacrylamide gel electrophoresis. (c) CNBr peptide analysis. These methods gave consistent and reproducible results, indicating a selective decrease of type I collagen in Fraser mouse skin as compared to control mouse skin. Metabolic labelling of skin organ cultures showed a decreased specific radioactivity of hydroxy[³H]proline in type I collagen of Fraser mouse skin. The concordant results of these experiments suggest a genetically determined alteration of interstitial collagen metabolism in the Fraser mutation apparently specifically concerning the expression of type I collagen gene(s).     

Interpretation of the electron microscopical appearance of collagen fibrils from corneal stroma

The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea.     

Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans

Several collagen genes have been isolated from the nematode Caenorhabditis elegans. The complete nucleotide sequences of two of these genes, col-1 and col-2, have been determined. These collagen genes differ from vertebrate collagen genes in that they contain only one or two introns, their triple-helical regions are interrupted by nonhelical amino acid sequences and they are smaller. A high degree of nucleotide and amino acid homology exists between col-1 and col-2. In particular, the regions around cysteines and lysines are most highly conserved. The C. elegans genome contains 50 or more collagen genes, the majority of which probably encode cuticle collagens; col-1 and col-2 apparently are members of this large family of cuticle collagen genes.     

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn²¹¹ residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved ²¹¹NDS N-glycosylation motif, but not other N-glycosylation sites (Asn²⁶⁰, Asn³⁷¹, and Asn³⁹⁴), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr⁴⁸⁴, Tyr⁵²⁰, Tyr⁷⁹², and Tyr⁷⁹⁷. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved ²¹¹NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.