Heilwig Rohde

Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix

Abstract Laminin from a mouse tumor basement membrane and fibronectin from human blood plasma were examined by electron microscopy using rotary shadowing and negative staining and by transmission scanning electron microscopy of unstained samples. Laminin was visualized as a rigid, asymmetric cross consisting of a long (77 nm) and three apparently identical short (36 nm) arms. The rod-like arms (diameter about 2 nm) terminated in globular units (diameter 5 to 7 nm). Additional globules were found near the terminal units in the short arms. A large pepsin-resistant fragment of laminin appeared as a rigid structure with three arms (length 26 nm, preferred angle 90 °), which presumably represented parts of the three short arms of laminin. Fibronectin could be visualized as two identical strands (length 61 nm, diameter about 2 nm), which did not reveal distinct globular units. These strands very likely comprised single peptide chains connected to each other at one end, enclosing a fixed angle of about 70 °. Electron microscopy also indicated a limited flexibility of the arms of both laminin and fibronectin, comparable to the stiffness of tropomyosin or DNA. The electron microscopic images of the shapes and dimensions of laminin, of fragments of laminin, and of fibronectin are consistent with the specific molecular weights and with the hydrodynamic properties determined in solution. The arms of fibronectin showed three distinct regions at which preferential bending occurred. These sites apparently correspond to flexible segments connecting more compact domains previously identified in biochemical studies. No sites of preferential bending were visible in the arms of laminin. Although laminin and fibronectin have some similar biological activities (binding of cells, collagen, glycosaminoglycans), the corresponding functional domains are differently arranged in the two molecules.

Radioimmunoassay for type III procollagen peptide and its application to human liver disease.

Abstract. A sensitive and specific radioimmunoassay was developed for the precursor‐specific peptide segment located at the amino end of bovine type III procollagen. Human material showed high cross‐reactivity in this assay. Two forms of human procollagen peptides were detected in body fluids. The larger peptide (45K) was found in serum and ascites, and resembled the whole precursor‐specific segment which is presumably released from human type III procollagen by a single enzymatic cleavage. The smaller peptide (10K) was found mainly in urine indicating that further degradation of circulating procollagen peptides is required prior to their passage through the kidney.

Sensitive radioimmunoassays for 7 S collagen and laminin: application to serum and tissue studies of basement membranes.

Abstract Radioimmunoassays were developed for the basement membrane components, 7 S collagen and fragments of the noncollagenous protein laminin, which allowed quantitative analysis of as little as 0.1–0.4 ng of these proteins. Similar materials could be detected by these assays in serum, in kidney digests, and in the medium of cell cultures obtained from mice and rats. Distinct changes in the amounts of antigen in serum and kidney were observed during aging and in mice inoculated with a basement membrane tumor.

Serum and Urine Analysis of the Aminoterminal Procollagen Peptide Type III by Radioimmunoassay with Antibody Fab Fragments

A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.

Radioimmunoassay for the aminoterminal peptide of procollagen pα1(I)-chain

Abstract Peptides derived from the aminoterminal portion of the pα1(I)-chain of calf and sheep procollagen were labeled with iodine-125. Despite changes in electrophoretic homogeneity after labeling, reaction of the labeled peptide with antisera to unlabeled peptide was retained. Antisera to procollagen or the isolated procollagen peptide showed high titers for the native peptide, a much weaker binding with the reduced and alkylated peptide and little or no reaction with collagen. Antisera to collagen showed strong binding with collagen and a weaker but distinct reaction with the procollagen peptide. Evidence was obtained that a minor contaminant of procollagen peptide was present in the acid-extracted collagen and that there were no shared antigenic determinants. Bovine serum and amniotic fluid contained 1–10 μg/ml reactive antigen. These results indicate that the labeled peptides can be used as a specific, sensitive and accurate assay for the amino-terminal portion of procollagen in biological samples.

Antigenic structure of the aminoterminal region in type I procollagen: Characterization of sequential and conformational determinants

Abstract Antibodies to calf or sheep skin procollagen, to its consitituent pα1(I) and pα2 chain or to peptide fragments from the aminoterminal region in pα1(I) were studied by radioimmune assays using various 125 0-labeled antigens. The highest antibody response was obtained against the globular region in pα1(I). The adjacent precursor-specific collagenous and non-helical domains may modulate the antigenicity of the globular determinants but are themselves only weak antigens. No antigenic determinants were detected in the pα2 chain. Antibodies to pα1(I) chain did not cross-react with pα2 or pα1(III) chains. Antisera against the globular procollagen peptide showed no difference between the peptide and procollagen indicating that the release of the globular region from pα1(I) by collagenase is not accompanied by large conformational alterations. Complete cross-reaction was observed between precursor-specific peptides obtained from calf or sheep procollagen. Reduction and S-carboxymethylation of the five disulfide bridges in the globular region largely destroyed antigenicity. However, a minor fraction of the antibodies against the native peptide could still react with the unfolded peptide. Determinants recognized in this reaction are apparently shared by the native and unfolded peptide and were stable towards trypsin. On the other hand, antibodies prepared against the reduced and alkylated procollagen peptide did not react with the native peptide. The data indicate that that globular regions in pα1(I) chain possesses both conformational and sequential determinants which differ considerably in their immunogenic capacity.

Genetic control and carrier and suppressor effects in the antibody response of mice to procollagen.

The response potential of inbred mouse strains against bovine type I procollagen and its separated structural domains, i.e., the globular procollagen peptide and the triple-helical collagen moiety, was compared by passive hemagglutination and radioimmune assays. Studies with congenic and recombinant lines and with backcross populations showed that the antibody response to procollagen peptide is under the control of a gene located in theIA, IB subregion of theH-2 complex. The strain distribution pattern of high response to the procollagen peptide is not identical to that of the high response to collagen. Both the procollagen peptide and procollagen are thymus-dependent antigens, since no response was observed in nude mice. Low response to procollagen peptide and/or collagen could be corrected in some, but not all mouse strains by using procollagen as immunogen. Strains which show low response against collagen but high response against procollagen were injected with collagen prior to a challenge with procollagen. This treatment reduced the antibody response to the procollagen peptide but not to collagen. Carrier and suppressor effects in the response to procollagen are apparently more complex than those observed in the response to synthetic peptides.

Pathobiochemie der Leberfibrose: Bedeutung der Prokollagenpeptide im Serum für Diagnose und Verlaufskontrolle

Fur Diagnose und Therapie einer Leberfibrose ware es Voraussetzung, ihre Aktivitat mit einem einfachen klinischen Test messen zu konnen. Wenn die Aktivitat einer Fibrose an der Syntheserate von Bindegewebssubstanzen gemessen wird, so wurden mehrere Parameter der Biosynthese von Kollagenen, Proteoglykanen und Glykoproteinen der interstitiellen Matrix in Frage kommen, die in Biopsien, Serum oder Urin gemessen werden konnen [1]. Ihre Bedeutung bedarf der Uberprufung in prospektiven klinischen Studien und mus an sorgfaltigen histologischen Verlaufsbeobachtungen gemessen werden, die unverandert die entscheidenden klinischen Informationen liefern.