We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.

Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1981.tb05690.x

A Network Model for the Organization of Type IV Collagen Molecules in Basement Membranes

Basement membranes are thin layers of a specialized extracellular matrix that form the supporting structure on which epithelial and endothelial cells grow, and that surround muscle and fat cells and the Schwann cells of peripheral nerves. One common denominator is that they are always in close apposition to cells, and it has been well demonstrated that basement membranes do not only provide a mechanical support and divide tissues into compartments, but also influence cellular behavior. The major molecular constituents of basement membranes are collagen IV, laminin-entactin/nidogen complexes, and proteoglycans. Collagen IV provides a scaffold for the other structural macromolecules by forming a network via interactions between specialized N- and C-terminal domains. Laminin-entactin/nidogen complexes self-associate into less-ordered aggregates. These two molecular assemblies appear to be interconnected, presumably via binding sites on the entactin/nidogen molecule. In addition, proteoglycans are anchored into the membrane by an unknown mechanism, providing clusters of negatively charged groups. Specialization of different basement membranes is achieved through the presence of tissue-specific isoforms of laminin and collagen IV and of particular proteoglycan populations, by differences in assembly between different membranes, and by the presence of accessory proteins in some specialized basement membranes. Many cellular responses to basement membrane proteins are mediated by members of the integrin class of transmembrane receptors. On the intracellular side some of these signals are transmitted to the cytoskeleton, and result in an influence on cellular behavior with respect to adhesion, shape, migration, proliferation, and differentiation. Phosphorylation of integrins plays a role in modulating their activity, and they may therefore be a part of a more complex signaling system.

Basement Membrane Proteins: Structure, Assembly, and Cellular Interactions

Four decades have passed since the first discovery of collagen IV by Kefalides in 1966. Since then collagen IV has been investigated extensively by a large number of research laboratories around the world. Advances in molecular genetics have resulted in identification of six evolutionary related mammalian genes encoding six different polypeptide chains of collagen IV. The genes are differentially expressed during the embryonic development, providing different tissues with specific collagen IV networks each having unique biochemical properties. Newly translated α-chains interact and assemble in the endoplasmic reticulum in a chain-specific fashion and form unique heterotrimers. Unlike most collagens, type IV collagen is an exclusive member of the basement membranes and through a complex inter- and intramolecular interactions form supramolecular networks that influence cell adhesion, migration, and differentiation. Collagen IV is directly involved in a number of genetic and acquired disease such as Alport's and Goodpasture's syndromes. Recent discoveries have also highlighted a new and direct role for collagen IV in the development of rare genetic diseases such as cerebral hemorrhage and porencephaly in infants and hemorrhagic stroke in adults. Years of intensive investigations have resulted in a vast body of information about the structure, function, and biology of collagen IV. In this review article, we will summarize essential findings on the structural and functional relationships of different collagen IV chains and their roles in health and disease.

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Mammalian collagen IV.

Specific antibodies to collagen type IV, laminin, and fibronectin were used to localise these proteins by indirect immunofluorescence in frozen sections of normal and fibrotic liver. In normal livers distinct staining was found in basement membranes of blood and lymph vessels, of bile ducts and ductules and around nerve axons. Positive reactions for type IV collagen and fibronectin were also observed in the perisinusoidal space, while hepatocytes and most of the interstitial matrix of portal fields remained unstained. Liver specimens obtained from patients with alcoholic liver disease (fatty liver, hepatitis or cirrhosis) and chronic active hepatitis showed a more intense reaction with the antibodies in the perisnusoidal space including now distinct staining for laminin. These patterns were particularly prominent at borders between fibrotic septa and remnants of parenchyma or pseudolobules. Strong reactions were also found for type IV collagen and fibronectin in the periportal interstitium and in large fibrotic areas. The findings support previous electron-microscopical and chemical evidence for increased basement membrane production in human liver fibrosis and demonstrate that this may involve different proteins and occur at different anatomical sites.

https://gut.bmj.com/content/gutjnl/21/1/63.full.pdf

Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2879%2981022-4

Juha Risteli

Papers

Identification of a new basement membrane collagen by the aid of a large fragment resistant to bacterial collagenase