http://www.jbc.org/content/254/19/9933.full.pdf

Laminin--a glycoprotein from basement membranes.

We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.

Basement Membrane Complexes with Biological Activity

The induction of polyarthritis in rats by intradermal immunisation with homologous or heterologous type II collagen incomplete or incomplete Freund's adjuvant was reported recently by Trentham et al. We have now produced a similar disease in certain strains of mice.

Immunisation against heterologous type II collagen induces arthritis in mice

Publisher Summary The chapter discusses the protein stability with emphasis on compact globular proteins representing a single cooperative system. All the small compact globular proteins represent cooperative systems; they exhibit an extreme cooperativity that integrates the whole of their structure into a single structural unit. The large proteins, to which fibrillar proteins are also related, do not present single cooperative systems, but are subdivided into definite cooperative subsystems—structural blocks or domains. The advances in studying the stability of complicated proteins are connected with two methodical achievements: (1) the appearance of the precise scanning microcalorimetric technique, which affords reliable information on the heat capacity function of proteins in a broad temperature range; and (2) realization of the fact that the complicated heat effect of disruption of a complex macromolecular structure can be analyzed thermodynamically. The thermodynamic specificity of collagen has been considered. The volume of globular proteins does not increase at denaturation but decreases, as seen from their ability to denature under high pressure. The results of calorimetric studies are discussed, presenting the specific melting enthalpy of various protein structures—globular proteins, double-stranded coiled coils, and triplestranded coiled coils. The practical importance of thermodynamic studies of protein stability—that is, its importance not only for understanding the principles of organization of these molecules, but just for obtaining structural information on the domain level is emphasized.

Stability of proteins. Proteins which do not present a single cooperative system

(First of Two Parts) COLLAGEN is the major macromolecule of most connective tissues, and it is widely believed that many human diseases, particularly chronic diseases, will be found to involve this protein either directly or indirectly. Since collagen biosynthesis was last reviewed in the Journal in 1972,1 much new information has been gained about the relatively complex pathway by which it is synthesized. We should like to consider this new information and some of its consequences in terms of the following questions: What is the structural information in the collagen molecule, and how does it determine the structure of the . . .

Biosynthesis of collagen and its disorders

Nidogen was purified from a mouse tumor basement membrane where it accounted for 2-3% of the total proteins. It was isolated as two forms (A and B) of a monomer (Mr = 80000) each consisting of a single polypeptide chain folded into a globular head connected to a small tail. The B form of the monomer was shown to be capable of aggregating into a nest-like structure (Mr greater than 250000). A smaller form (Mr = 45000) was observed in some of the extracts. The amino acid composition of nidogen was different to that of other basement membrane proteins. It contained about 10% carbohydrate, with N-linked and O-linked oligosaccharide chains in similar proportions. Isoelectrofocussing demonstrated a limited heterogeneity of nidogen with pI in the range 6.5 - 7. Monomeric nidogen failed to interact with other basement membrane components and heparin. Aggregation could be induced by limited proteolysis and was reversed by detergents or high salt concentrations. Together with the observation that most of the nidogen could be solubilized only after destroying the collagenous matrix, the data indicate that aggregation of nidogen reflects an activity involved in matrix assembly. Specific antibodies raised against nidogen did not distinguish between the monomeric and aggregated form of the protein but showed that the fragment was antigenically deficient. These antibodies did not cross-react with collagen type IV, laminin, entactin and heparansulfate proteoglycan. Immunofluorescence staining and absorption studies demonstrated that nidogen is a common component of authentic basement membranes. Larger forms of nidogen (Mr about 100000 and 150000) were found in organ cultures of Reichert's membrane suggesting that it is synthesized in precursor forms.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07849.x

Nidogen: a new, self-aggregating basement membrane protein.

Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.

https://rupress.org/jem/article-pdf/166/2/419/1097192/419.pdf

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Extraction of fetal bovine skin at neutral pH and in the presence of protease inhibitors solubilized substantial amounts of type I and type type III collagen and Type III procollagen. Type I and Type III collagen were separated from each other by salt precipitation, and DEAE-cellulose chromatography was used to separate collagen from procollagen. The main chain constituents in type III collagen and procollagen were disulfide-bonded gamma and pgamma components, respectively. Amino acid composition, cross striation banding as observed using electron microscopy, cyanogen bromide peptide patterns in disc electrophoresis gels and resistance of the disulfide regions to pepsin digestion indicated a close similarity to previously described insoluble type III collagen, which was solubilized by limited pepsin digestion. Electron microscopy of long-spacing-segment crystallites and evidence for an extended form of the disulfide-bonded cyanogen bromide peptide suggested that neutral-salt-soluble type III collagon is longer at its C-terminal end by about 10 to 20 amino acid residues than pepsin-treated material. A small elongation was also indicated in the N-terminal portion of the molecule. Procollagen has an additional N-terminal extension with a length of about 160 A, but no difference was observed between collagen and procollagen at their C-terminal ends.

Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin.

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr= 150000) and, in addition, two defined fragments (Mr= 130000 and 100000). Smaller fragments (Mr= 80000 and 40000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr= 80000 fragment. Additional epitopes were found on intact nidogen and the Mr= 130000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr= 80000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1986.tb09605.x

Hans Nowack

Papers

Nidogen: a new, self-aggregating basement membrane protein.

Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin.

Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane