Shirley Ayad

Isolation and characterisation of an unusual collagen from hyaline cartilage and intervertebral disc

The major collagen of hyaline cartilage is type II collagen, consisting of 3 identical cul(ll) chains [1,2]. However, additional minor collagenous components have been observed (but not identified) in chick scleral cartilage [3] and chick embryonic limb cartilage [4], and the aB (or al(V)) chain of type V collagen was reported in epiphyseal cartilage [S]. In [6], 3 collagen o-sized chains from human hyaline cartilage were isolated and partially characterised. Two of these chains designated la and 2cu, were very similar to the cwB (al(V)) and cuA ((u2(V)) chains, respectively, of type V collagen but were shown to be genetically distinct by several physicochemical criteria. The third chain 3a, however, resembled the ol(lI) chain of type II collagen and could be a post-translational modification of this chain. We have studied the collagens of bovine nasal cartilage and human intervertebral disc and have identified the lo, 20 and 3a chains in addition to type II collagen in both tissues. However, we have never observed the cut(V) chain which we believe originates only from contaminating perichondrium. We have also isolated an unusual collagen which is different from any other collagen reported in cartilage or disc. This report describes its isolation and characterisation.

Incidence of antibodies to native and denatured cartilage collagens (types II, IX, and XI) and to type I collagen in rheumatoid arthritis.

The frequencies of antibodies to the cartilage type IX and XI collagens and to type I collagen were determined in 188 patients with rheumatoid arthritis, of whom 76 were positive for antibodies to native type II collagen. A higher proportion of patients with antibodies to native type II collagen had antibodies to these other collagens, but about one third of patients without antibodies to native type II collagen had antibodies to one or more denatured collagens. The patterns of antibodies present in individual sera suggested that there was a selective response to the collagens in an individual patient. The incidence of patients having antibodies to these native and denatured collagens in a random group of patients with rheumatoid arthritis was calculated.

Experimental osteoarthritic articular cartilage is enriched in guanidine-soluble type VI collagen

Experimental osteoarthritis was surgically induced in the right knee joint of dogs; the left knee served as a control. Articular cartilage was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate, pH 6.0, containing proteinase inhibitors and the proteins purified by associative CsCl density gradient centrifugation. Equal quantities of protein were electrophoresed in agarose-acrylamide gradient gels and the high molecular weight type VI collagen bands detected in immunoblots with a polyclonal antiserum. Type VI collagen bands between 185 and 220 kDa were evident in the pathological specimens of dogs sacrificed 3, 5, and 7 months after surgery and were either absent or only very weakly visible in the controls. These results demonstrate that experimental osteoarthritic cartilage is enriched in 4 M guanidine-soluble type VI collagen.

Characterisation of another short-chain disulphide-bonded collagen from cartilage, vitreous and intervertebral disc

Cartilage is an avascular gel-like tissue having a collagenous meshwork made up mainly of a type of collagen (type II) present only in tissues of a similar nature such as vitreous and intervertebral disc [l-3]. Minor collagens la2a3cr [4] and disulphide-bonded phosphate-soluble collagens [S-7] have been demonstrated in cartilage and intervertebral disc which are not present in other dissimilar tissues. At present, the configuration of the phosphate soluble collagens is not clearly established. Here we describe the isolation and character&ration of a second such collagen from cartihage, vitreous and intervertebral discs.

Isolation and native characterization of cysteine-rich collagens from bovine placental tissues and uterus and their relationship to types IV and V collagens.

A simplified procedure for the fractionation and purification of different collagen types from various tissues is described which is particularly efficient in separating type-V from type-IV collagen, and highmol.-wt. (HMW) aggregates from 7 S collagen. Uterus and maternal villi contain 2 forms of type-V collagen -{α1(V)}2α2(V) and {α1(V)2α2(V)α3(V)}—which have been separated on DEAE-cellulose. Uterus however appears to be the richest source of both HMW aggregates and the {α1(V)2α2(V)α3(V)} collagen, and a probable relationship between these collagens is discussed.

Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix.

Correspondence to: ProfessorM E Grant, School of Biological Sciences, Stopford Building, University of Manchester, Oxford Road, Manchester Ml 3 9PT, United Kingdom. The collagens constitute a family of proteins that are assembled into a variety of supramolecular structures in extracellular matrices. Research conducted over the last 20 years has led to the identification of at least 16 genetically distinct collagen types encoded by over 30 different genes. The structure, function and distribution of these collagens are still the subject of intense investigation, but on the basis of their known gene structure and amino acid sequences it is possible to classify them into different groups. The most studied group is that comprising the collagens capable of forming classical fibrous structures (types I, II, III, V and XI), whose main function is to resist tensile stresses exerted on tissues. Another class of collagenous molecules includes types IX, XII and XIV which are believed to be associated with the fibrous collagens and have been assigned the name of Fibril Associated Collagens with Interrupted Triple-helices (FACIT). Collagen types VIII and X appear to form another class of matrix proteins for they exhibit remarkable homology at the levels of gene organisation and amino acid sequence and have the ability to form regular hexagonal lattice structures. A detailed review of all collagen types is published elsewhere. 1 Individual collagen types are rarely found in isolation in extracellular matrices and generally occur in conjunction with several others. For example, collagen types I, III, V and VI are widely distributed in connective tissues such as skin, tendon, aorta, gut etc. In contrast, collagen IV is localised exclusively to basement membranes and collagen VII has only been described in the anchoring fibrils underlying the lamina densa of many epithelia. During evolution a separate group of collagens has arisen to provide cartilaginous tissues with their own distinct macromolecular organisation. Here we find the cartilage-specific collagens (types II, IX, X and XI) together with the more ubiquitous collagen type VI, and possibly types XII and XIV (fig 1). There is still much to be discovered about the role of these collagens in cartilage and especially about their interactions with the highly hydrated proteoglycans of the cartilage matrix. The collagens must function not only to resist tensile forces and to distribute load on bones articulating in cartilaginous and synovial joints but also to mediate the growth and development of the skeleton. The critical role of collagen II in maintaining cartilage integrity has been demonstrated by studies on certain heritable chondrodysplasias, whereby aberrant collagen II synthesis gives rise to a spectrum of clinical symptoms ranging from mild dwarfism, myopia and secondary osteoarthritis (OA) in some forms of spondyloepiphyseal dysplasia (SED) to several lethal forms of achondroplasia.2 It is clear that the cartilage collagens are crucial determinants of the mechanical properties of all articulating joints and any disturbance of the complex collagenous

1 Alpha 2 alpha 3 alpha collagen is arthritogenic.

Native 1 alpha 2 alpha 3 alpha collagen (500 micrograms per rat) was both immunogenic and arthritogenic in Alderley Park rats (46% developed arthritis) but only immunogenic in Sprague-Dawley rats. Conversely, native type II collagen (500 micrograms per rat) was immunogenic and arthritogenic in both strains (64% arthritic in Alderley Park strain, 57% arthritic in Sprague-Dawley strain). The inflammatory polyarthritis induced by 1 alpha 2 alpha 3 alpha collagen was similar to that produced by native type II collagen in clinical appearance, time of onset, and histology. Antibodies raised to native bovine type II collagen cross-reacted with native 1 alpha 2 alpha 3 alpha collagen and vice versa. Thus the minor collagen component of cartilage, the 1 alpha 2 alpha 3 alpha collagen, as well as the major collagen component, type II collagen, are immunogenic and arthritogenic in the rat, with strain differences.

Extraction and characterisation of the intact form of bovine vitreous type IX collagen

We provide the first biochemical characterisation of intact type IX collagen extracted from bovine vitreous. It possesses a shortened alpha 1(IX) chain (M(r) 64K) compared to its cartilage counterpart (M(r) 84K). All the vitreous type IX collagen is in a proteoglycan form, its glycosaminoglycan constituent being a chondroitin/dermatan sulphate component of M(r) 15-60K attached to the alpha 2(IX) chain. This contrasts with previous findings in chick vitreous where a very long glycosaminoglycan chain of M(r) approximately 350K was demonstrated.

The morphological features of bovine meshwork cells in vitro and their synthetic activities

The morphological characteristics of 3rd-passage cultured bovine meshwork cells were investigated, as were some of their synthetic activities. Growing meshwork cells had the ultrastructural characteristics of metabolically active cells whereas postconfluent cells formed gap junctions at their lateral borders and were closer in their fine structure to meshwork cells in vivo. The incorporation of3H proline and3H glucosamine was significantly greater in growing than postconfluent cultures. The normal bovine outflow system was rich in type I collagen but type V collagen and fibronectin was also evident. The 3rd-passage cells were shown to synthesize type I collagen, type V collagen and fibronectin. However, ultrastructural studies of the extracellular matrix which was produced in vitro demonstrated the presence of small fibrils with no distinctive banding pattern, unlike the larger collagen fibrils with a 640 Å banding pattern seen in vivo.

Partial characterization of type X collagen from bovine growth-plate cartilage Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth‐plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [α1(X)]3 form in vivo, although the procollagen [proα1(X)]3 form can also be detected. The bovine proα1 (X) and α1(X) chains have M r, values identical to the corresponding chick species (M r 59 000 and 49 000). However, the pepsinized α1(X)p chains (M r 47 000) are larger than those of the chick (M r 45 000), and the bovine collagen type X is further distinguished by being disulphide‐bonded within the triple‐helical domain.