A modern viewpoint on structure and evolution of collagens. I. Fibrillar collagens

This review summarizes current data on structure of the most representative group of the collagen family--fibrillar collagens. Attention has been focused on structural organization of individual domains and their functional role in the hierarchical stacking of alpha-chains of collagens. There is presented characteristics of the main stages of biosynthesis and of supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene rearrangements, and exon shuffling in evolution of collagen genes is discussed.     

The collagens of hydra provide insight into the evolution of metazoan extracellular matrices

A collagen-based extracellular matrix is one defining feature of all Metazoa. The thick sheet-like extracellular matrix (mesoglia) of the diploblast, hydra, has characteristics of both a basement membrane and an interstitial matrix. Several genes associated with mesoglea have been cloned including a basement membrane and fibrillar collagen and an A and B chain of laminin. Here we report the characterization of a further three fibrillar collagen genes (Hcol2, Hcol3, and Hcol5) and the partial sequence of a collagen gene with a unique structural organization consisting of multiple von Willebrand factor A domains interspersed with interrupted collagenous triple helices (Hcol6) from Hydra vulgaris. Hcol2 and -5 have major collagenous domains of classical length ( approximately 1020 amino acid residues), whereas the equivalent domain in Hcol3 is shorter (969 residues). The N-propeptide of Hcol2 contains a whey acid protein four-cysteine repeat (WAP) domain, and the equivalent domain of Hcol3 contains two WAP and two von Willebrand factor A domains. Phylogenetic analyses reveal that the hydra fibrillar collagen genes form a distinct clade that appears related to the protostome/deuterostome A clade of fibrillar collagens. Data base searches reveal Hcol2, -5, and -6 are highly conserved in Hydra magnipapillata, which also provided preliminary evidence for the expression of a B-clade fibrillar collagen. All four of the H. vulgaris collagens are expressed specifically by the ectoderm. The expression pattern for Hcol2 is similar to that previously reported for Hcol1 (Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S., Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000) Development 127, 4669-4680) but distinct from the pattern shared by Hcol3 and Hcol5. The characterization of multiple collagen genes in relatively simple diploblastic organisms provides new insights into the molecular evolution of collagens and the origins of the collagen-based extracellular matrix found throughout the multicellular animal kingdom.     

Demosponge and sea anemone fibrillar collagen diversity reveals the early emergence of A/C clades and the maintenance of the modular structure of type V/XI collagens from sponge to human

Collagens are often considered a metazoan hallmark, with the fibril-forming fibrillar collagens present from sponges to human. From evolutionary studies, three fibrillar collagen clades (named A, B, and C) have been defined and shown to be present in mammals, whereas the emergence of the A and B clades predates the protostome/deuterostome split. Moreover, several C clade fibrillar collagen chains are present in some invertebrate deuterostome genomes but not in protostomes whose genomes have been sequenced. The newly sequenced genomes of the choanoflagellate Monosiga brevicollis, the demosponge Amphimedon queenslandica, and the cnidarians Hydra magnipapillata (Hydra) and Nematostella vectensis (sea anemone) allow us to have a better understanding of the origin and evolution of fibrillar collagens. Analysis of these genomes suggests that an ancestral fibrillar collagen gene arose at the dawn of the Metazoa, before the divergence of sponge and eumetazoan lineages. The duplication events leading to the formation of the three fibrillar collagen clades (A, B, and C) occurred before the eumetazoan radiation. Interestingly, only the B clade fibrillar collagens preserved their characteristic modular structure from sponge to human. This observation is compatible with the suggested primordial function of type V/XI fibrillar collagens in the initiation of the formation of the collagen fibrils.     

FACIT collagens: diverse molecular bridges in extracellular matrices

The collagens form a large family of proteins. Collagen fibrils, composed of staggered arrays of fibrillar collagen molecules (types I, II, III, V and XI), provide a supporting scaffold for extracellular matrices of connective tissues. The non-fibrillar collagens are less abundant than the fibrillar collagens, but it is becoming clear that they have important functions in the matrix. Recently, a group with unique structural characteristics has been defined and named the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) group. There is evidence that these collagens may serve as molecular bridges that are important for the organization and stability of extracellular matrices.     

Collagen XXVII is developmentally regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.     

Current viewpoint on structure and on evolution of collagens. II. Fibril-associated collagens

Fibril-associated collagens (FACITs) form one of subfamilies included in family of collagens. Being minor components of connective tissue of multicellular animals, FACITs play an important role in structurization of extracellular matrix whose peculiarities determine essential intertissue differences. FACITs participate in regulation of sizes of banded collagen fibrils as well as are connecting links between various components extracellular matrix and cells in different tissues. Functional characteristics of FACIT molecules are determined by peculiarities of structural organization of their α-chains (breakdowns in collagenous domains and module structure of N-terminal noncollagenous sites), trimeric molecules (domains of trimerization) and supramolecular assemblies (mainly association with banded collagen fibrils and the inability to form homopolymeric supramolecular aggregates). The problem of evolution of this group of collagen molecules is also discussed. A hypothetical model of structural changes leading to formation of the FACIT subfamily is proposed.     

Dermis collagens: beyond their structural properties

The extracellular matrix is a complex network composed of macromolecules such as collagens, proteoglycans and elastin that strongly interact with each other and with cells to maintain the structural integrity of many tissues. These interactions also sustain important cell programs such as migration, proliferation, differentiation and apoptosis. The skin, and more specifically the dermis, contains an extreme diversity of macromolecules that reflects the importance of the composition and organization of the matrix components in providing physical properties and function of the tissues. The most abundant matrix components are the collagens that form a super-family of 27 different members which are divided into different subgroups. The fibrillar collagens, types I, III and V, the FACIT collagens, types XII, XIV and XVI, and collagen VI are all expressed in the collagen-rich dermis. Although the structural features of these collagens are now well characterized, their functions remain elusive. Mutations in human collagen genes give rise to numerous connective tissue diseases including dermis disorders. For example, clinical manifestations in the classical Elhers-Danlos syndrome caused by collagen V gene mutations occur predominantly in the dermis. However, the genotype-phenotype relationship is not clearly established as well as the relation between the distribution and the function of the collagens in dermis. There is no doubt that the ongoing and future work using in vivo approaches will provide new cues regarding the function of collagens in dermis.     

Molecular and biochemical aspects of nematode collagens.

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens also are cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the proteins.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium 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.     

Fibrillar collagen: The key to vertebrate evolution? A tale of molecular incest

Fibril-forming (fibrillar) collagens are extracellular matrix proteins conserved in all multicellular animals. Vertebrate members of the fibrillar collagen family are essential for the formation of bone and teeth, tissues that characterise vertebrates. The potential role played by fibrillar collagens in vertebrate evolution has not been considered previously largely because the family has been around since the sponge and it was unclear precisely how and when those particular members now found in vertebrates first arose. We present evidence that the classical vertebrate fibrillar collagens share a single common ancestor that arose at the very dawn of the vertebrate world and prior to the associated genome duplication events. Furthermore, we present a model, 'molecular incest', that not only accounts for the characteristics of the modern day vertebrate fibrillar collagen family but demonstrates the specific effects genome or gene duplications may have on the evolution of multimeric proteins in general.     

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.     

Degradation of monomeric and fibrillar type III collagens by human skin collagenase. Kinetic constants using different animal substrates

Human skin collagenase activity was examined against type III collagens, in both soluble and fibrillar form, from different animal species. In either form, human, dog, and cat type III were degraded 10- to 30-fold faster than was that from guinea pig and nearly 100-fold more readily than chick type III. These differences in susceptibility were mirrored by essentially identical differences in the rate of trypsin cleavage of the same substrates. Human, dog, and cat type III were cleaved most rapidly by trypsin, guinea pig III more slowly, and chick III was completely resistant to the serine protease. Arrhenius plots, relating enzyme activity to temperature, revealed differences in the various type III substrates consistent with their collagenase and trypsin susceptibilities. Human, dog, and cat type III collagens yielded nonlinear plots, with accompanying activation energies which decreased at temperatures above 26 degrees C; guinea pig type III displayed a plot which deviated only slightly from linearity while the plot for chick type III was completely linear. These data strongly suggest that type III collagens display substantial variability in the stability of the helix at or near the collagenase cleavage site. The susceptibility of these type III substrates as reconstituted fibrils was also examined. The relative rates of degradation of these substrates by collagenase, and by trypsin, were the same as those observed in solution. The absolute rates of degradation of collagen in fibrillar form, however, were massively lower than predicted by extrapolation from solution values. This reduction in rate is even greater for type III than for type I collagens. Thus, whereas in solution type III substrates are cleaved much faster than type I collagens, in fibrillar form these differences are less than 2-fold. These data, together with values for activation energies and deuterium isotope effects on type III fibrillar substrates, reinforce the concept that helical integrity near the collagenase cleavage site is a major specifier of the rate of collagenase activity. Furthermore, the data suggest that the exclusion of water accompanying the tight packing of monomers into fibrils presents a major energy barrier to collagenase activity, which is particularly large for type III collagen.     

Short review. Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Isolation and biochemical characterization of collagens from seaweed pipefish, Syngnathus schlegeli

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the seaweed pipefish (Syngnathus schlegeli) and partially characterized. The amount of collagens isolated in the subsequent treatments was 5.5% of ASC and 33.2% PSC on the basis of lyophilized pipefish body weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the collagens might be classified as type I collagens, containing α1 and α2 chain. The imino acid content of collagen from pipefish was lower than those of mammalian collagens as also were the denaturation temperatures (Td) of collagens were 34.8°C and 35.1°C, respectively. This study shows that there is a possibility to use pipefish collagen as the alternative source of collagen from industrial purposes and subsequently it may evaluate the economical value of the seaweed pipefish.     

Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding,fibrillogenesis, cross-linking,and degradation

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.     

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.     

Tracing the evolution of vertebrate fibrillar collagens from an ancestral alpha chain.

From considerations of gene structure, phylogenetic analysis, modular organisation of related proteins and fibril shapes, we suggest a model for the evolution of contemporary vertebrate fibrillar collagens from a common ancestral alpha chain.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.