Making recombinant extracellular matrix proteins

A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.     

Immunocytochemical demonstration of two vitamin D-dependent calcium-binding proteins in mammalian kidney

Distribution of vitamin D-dependent calcium-binding proteins (CaBPs) were studied in four mammalian species using monospecific antibodies raised against chick duodenal CaBP (D-CaBP), human cerebellar CaBP (L-CaBP), and rat duodenal CaBP (S-CaBP). The immunoperoxidase technique of unlabelled antibodies was employed. The distribution of D-CaBP/L-CaBP was identical in all the species studied except for the monkey. In the rat, pig, and human nephrons, D-CaBP/L-CaBP was seen in the cytoplasm of the cells of the distal convoluted tubules, initial segments of the collecting ducts and interspersed cells of the collecting ducts. Proximal convoluted tubules, glomeruli and maculae densae were negative. In the monkey, in addition to the cells of the distal convoluted tubules, the cells along the entire length of the collecting ducts were also strongly positive. S-CaBP was found to be species-specific, and hence positive results were obtained only in the rat nephron. The strongest positive reaction for S-CaBP was seen in the cells of the distal convoluted tubules. These same cells were also positive for D-CaBP/L-CaBP. S-CaBP was also detected in the cells of the thick ascending limb of the loop of Henle, along the entire length of the collecting ducts and in smaller amounts in cells of the macula densa. Intracellularly the S-CaBP was present only in the apical cytoplasm of positive cells. D-CaBP/L-CaBP stained the entire cytoplasm but the staining in the apical cytoplasm was denser.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

Immunocytochemical demonstration of lysosomal matrix vesicles in the arterial wall of the rat

Following necrobiosis of the smooth muscle cells (SMC) of the vessel wall, lysosomes are still able to live for a time in the extracellular space. Here they are known as lysosomal matrix vesicles (MV). Their lysosomal origin can be confirmed by the immunocytochemical demonstration of beta-N-acetylglucosaminidase (beta-NAG) in extracellular MV. A positive reaction to the enzyme-cytochemical test for acid phosphatase establishes that these lysosomal MV are enzymatically active. The role of the lysosomal MV in the pathogenesis of vascular diseases is seen in an uncontrolled, locally limited destruction and alteration of the intercellular substance.     

Immunocytochemical demonstration of lysosomal matrix vesicles in the arterial wall of the rat

Summary Following necrobiosis of the smooth muscle cells (SMC) of the vessel wall, lysosomes are still able to live for a time in the extracellular space. Here they are known as lysosomal matrix vesicles (MV). Their lysosomal origin can be confirmed by the immunocytochemical demonstration of -N-acetylglucosaminidase (-NAG) in extracellular MV. A positive reaction to the enzyme-cytochemical test for acid phosphatase establishes that these lysosomal MV are enzymatically active. The role of the lysosomal MV in the pathogenesis of vascular diseases is seen, in an uncontrolled, locally limited destruction and alteration of the intercellular substance.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Binding of extracellular matrix proteins by lactobacilli

Ten gut and ten vaginalLactobacillus strains were investigated for their ability to bind type I collagen (Cn-I) and four selected gut lactobacilli were investigated for their binding to other extracellular matrix (ECM) molecules. Immobilized Cn-I (100 mg/L) in wells of microtitre plates was bound by all 10 autoaggregating vaginal strains and by 3 strains of gut lactobacilli from piglets in the range ofA ₅₇₀ readings 0.114–1.806.L. acidophilus strain SV31 was much more adherent than the rest of strains. All four gut lactobacilli tested for binding to other ECM molecules displayed good binding to porcine fibronectin and heparin and some of them bound weakly to fetuin and porcine mucin. No binding of these strains was observed to bovine mucin, bovine fibrinogen and bovine lactoferrin.     

Common structural motifs in proteins of the extracellular matrix

Proteins of the extracellular matrix are composed of many structurally and often functionally different autonomous domains which frequently occur as modular units in several different extracellular matrix proteins, but also in proteins of different origin. Some domains serve related assembly functions in different proteins but for domains involved in cell attachment and other cellular activities only a few generalizations are possible.     

Immunolocalization of stress proteins and extracellular matrix proteins in the rat tibia

Stress proteins (heat shock proteins [hsps]) serve a number of protective functions, including protection from apoptosis and acting as chaperones during protein biosynthesis. For example, hsp 27 has been defined as a chaperone for the G3 domain of aggrecan, while hsp 47 is the chaperone for type I collagen. Separate cytoprotective roles for hsp 27 and hsp 70 have been demonstrated. The aim of this study was to define the expression of hsps in osteoblastic and chondrocytic cells of the growing rat long bone in relationship to the immunohistochemical localization of aggrecan, type I collagen and the presence of fragmented DNA that defines apoptotic events. Tibiae were harvested from Fisher 344 rats (n=6) and fixed in 10% buffered formalin. Samples were decalcified in 10% EDTA, bisected, and processed for histologic examination. Sections (5 mm) were immunohistochemically stained using a streptavidin-biotin detection method. Co-localization of hsps with apoptosis was achieved using the TUNEL procedure. In the rat tibia growth plate, aggrecan was generally distributed throughout cartilage and chondrocytes. However, hsp 27 expression was observed only in the lower hypertrophic chondrocytes. hsp27 was present in osteoblasts lining newly formed bone. hsp 47 staining was also prominent within these osteoblasts where collagen type I immunolocalization occurred. The inducible form of hsp 70 was localized to the osteoblastic cells lining new bone in the primary spongiosa. In cartilage, DNA fragmentation was restricted to the hypertrophic, hsp27-positive, chondrocytes. In contrast, DNA fragmentation was not co-localized with hsp27-positive osteoblastic cells of the primary spongiosa, although occasional apoptotic cells were identified. These results indicate that apoptosis is a mechanism by which hypertrophic chondrocytes are eliminated from cartilage prior to calcification, but that other mechanisms are also likely to be involved. They also suggest that hsps have cytoprotective and biosynthetic functions within osteoblasts and chondrocytes, but apoptotic signals may override these effects in some instances, resulting in apoptosis.     

Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases

The proteolytic activity of four hemorrhagic metalloproteinases (Ht-a, c, d, and e) isolated from the venom of the Western diamondback rattlesnake (Crotalus atrox) was investigated using isolated extracellular matrix (ECM) proteins. We determined that all of the proteinases are capable of cleaving fibronectin, laminin, type IV collagen, nidogen (entactin), and gelatins. However, none of the proteinases were proteolytic against the interstitial collagen types I and III or type V collagen. With all of the substrates listed above Ht-c and Ht-d produced identical digestion patterns, as would be expected for these isoenzymes. With fibronectin, Ht-a produces a different ratio of products from Ht-c and Ht-d, while Ht-e produces a unique pattern of digestion. Ht-e and Ht-a produced nonidentical patterns with the laminin/nidogen preparation although some similarity was shared between them as well as with the Ht-c/d digestion pattern. Similar results were also observed for these proteinases with nidogen 150 as the substrate. The type IV collagen digestion patterns by Ht-e and Ht-a were similar to the pattern observed with Ht-c/d but differed by two bands. The digestion patterns of the three gelatins produced by the proteinases show differences between Ht-c and Ht-d when compared to Ht-e and Ht-a. This investigation clearly shows that several of the ECM proteins are efficiently digested by these toxins. The proteinases have some digestion sites in common but show differing specificities. In addition, the range of ECM proteins digested by these hemorrhagic proteinases is nearly identical to that demonstrated by the ECM proteinase stromelysin (MMP-3). From these data, and the knowledge of the roles these ECM proteins have in maintaining basement membrane structural/functional integrity, one can envision that the degradation of these ECM proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites.     

Selective proteolysis by matrix metalloproteinases of photo-oxidised dermal extracellular matrix proteins

Photodamage in chronically sun-exposed skin manifests clinically as deep wrinkles and histologically as extensive remodelling of the dermal extracellular matrix (ECM) and in particular, the elastic fibre system. We have shown previously that loss of fibrillin microfibrils, a key elastic fibre component, is a hallmark of early photodamage and that these ECM assemblies are susceptible in vitro to physiologically attainable doses of ultraviolet radiation (UVR). Here, we test the hypotheses that UVR-mediated photo-oxidation is the primary driver of fibrillin microfibril and fibronectin degradation and that prior UVR exposure will enhance the subsequent proteolytic activity of UVR-upregulated matrix metalloproteinases (MMPs).We confirmed that UVB (280-315 nm) irradiation in vitro induced structural changes to both fibrillin microfibrils and fibronectin and these changes were largely reactive oxygen species (ROS)-driven, with increased ROS lifetime (D₂O) enhancing protein damage and depleted O₂ conditions abrogating it. Furthermore, we show that although exposure to UVR alone increased microfibril structural heterogeneity, exposure to purified MMPs (1, −3, −7 and − 9) alone had minimal effect on microfibril bead-to-bead periodicity; however, microfibril suspensions exposed to UVR and then MMPs were more structurally homogenous. In contrast, the susceptibly of fibronectin to proteases was unaffected by prior UVR exposure. These observations suggest that both direct photon absorption and indirect production of ROS are important mediators of ECM remodelling in photodamage. We also show that fibrillin microfibrils are relatively resistant to proteolysis by MMPs −1, −3, −7 and − 9 but that these MMPs may selectively remove damaged microfibril assemblies. These latter observations have implications for predicting the mechanisms of tissue remodelling and targeted repair.     

Interaction of Helicobacter pylori with extracellular matrix proteins

Binding of 11 isolates of the gastroduodenal pathogen Helicobacter pylori from three geographical regions to extracellular matrix proteins was investigated. None of the strains bound soluble proteins, but a proportion (27%) bound immobilized laminin, fibronectin, and types I and V collagens. Microaerobic conditions were required to demonstrate reproducible binding. Contrary to previous reports, interstrain variation in the pattern of binding to various proteins was observed, possibly reflecting pathogenic or virulence differences between strains. Binding was strongest to laminin. Purified lipopolysaccharide completely inhibited the binding of H. pylori to laminin indicating that this bacterial surface molecule is involved in the adhesion process.     

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.     

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.     

Domain structure and organisation in extracellular matrix proteins.

Extracellular matrix (ECM) proteins are large modular molecules built up from a limited set of modules, or domains. The basic folds of many domains have now been determined by crystallography or NMR spectroscopy. Recent structures of domain pairs and larger tandem arrays, as well as of oligomerisation domains, have begun to reveal the principles underlying the higher order architecture of ECM proteins. Structural information, coupled with site-directed mutagenesis, has been instrumental in showing how adjacent domains can co-operate in ligand binding. Very recently, the first heterotypic ECM protein complexes have become available. Here, we review the advances of the last 5 years in understanding ECM protein structure, with special emphasis on those structures that have given insight into the biological functions of ECM proteins.     

Bacterial proteins binding to the mammalian extracellular matrix

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.     

Vascular wall extracellular matrix proteins and vascular diseases

Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension.     

Immunocytochemical demonstration of rod-opsin,S-antigen,and neuron-specific proteins in the human pineal gland

Summary The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%–5%; S-antigen immunoreaction: approximately 5%–10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.