Rupert Timpl

A new nomenclature for the laminins

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

Macromolecular organization of basement membranes

A considerable variety of basement membrane components, including in particular more than ten laminin isoforms and their novel alpha chains (alpha3, alpha4 and alpha5), has been characterized in recent studies. The functional properties of these components are increasingly being analyzed by recombinant technologies and by structural studies at atomic resolution, techniques which led to the elucidation of the nidogen-binding epitope on the laminin gamma1 chain. Novel insights into functions of basement membrane components have been obtained from gene-targeting experiments and studies of mutated genes identified in inherited disorders.

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival‐potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity‐purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin‐binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell‐binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod‐like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin‐binding domain of laminin is therefore responsible for its effects on neurons.

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.

Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding.

Endostatin, produced as recombinant protein in human 293‐EBNA cells, inhibits the migration of human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor (VEGF) in a dose‐dependent manner and prevents the subcutaneous growth of human renal cell carcinomas in nude mice at concentrations and in doses that are from 1000‐ to 100 000‐fold lower than those previously reported. The inhibition of migration is not affected by mutations which eliminate Zn or heparin binding and inhibition of tumor growth does not depend on Zn binding. The results of the migration assays suggest that endostatin causes a block at one or more steps in VEGF‐induced migration, while VEGF in turn can cause a block of the inhibition by endostatin of VEGF‐induced migration of HUVECs.

Arg-Gly-Asp constrained within cyclic pentapoptides Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1

Cyclic Arg‐Gly‐Asp‐Phe‐Val peptides with either D‐Phe or D‐Val residues were 20‐ to more than 100‐fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly‐Arg‐Gly‐Asp‐Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding or platelet receptor αIIbβ3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all‐trans conformation with a βII′ and γ turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity or inhibition.

Role of laminin a chain in the development of epithelial cell polarity

Kidney organ culture was used to study the conversion of embryonic mesenchymal cells into a polarized, differentiated kidney epithelium. We examined the expression of laminin, a basement membrane glycoprotein, during this conversion. The B chains of laminin were constitutively expressed, whereas the appearance of the A chain of laminin was dependent on embryonic induction and coincided with the onset of cell polarization. Antisera against the carboxy-terminal end of laminin inhibited polarization but did not affect the developmental events that precede polarization. Antisera against N-terminal parts of laminin failed to inhibit morphogenesis. Since the fragments at the carboxy-terminal end contain parts of the A chain, we suggest that the appearance of this chain is fundamental for initiation of cell polarity.

Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis.

Abstract Collagen α-chains and collagen peptides exhibit a lower electrophoretic mobility in SDS-polyacrylamide electrophoresis than globular proteins of the same size. Slight but significant differences were also noted between the α1- and the α2-chains and corresponding peptides. A more rigid structure in the polypeptide chains of collagen due to the high imino acid content was considered as the reason for these deviations. Peptides obtained from one chain, however, showed a strict linear relationship between the electrophoretic mobility and the logarithm of their molecular weight. Within these limits this method may be well suited for molecular weight estimations. This technique has also a high resolving power and should serve as a valuable analytical tool in studies on the primary structure of collagen.

Binding of the G domains of laminin α1 and α2 chains and perlecan to heparin, sulfatides, α‐dystroglycan and several extracellular matrix proteins

The C‐terminal G domain of the mouse laminin α2 chain consists of five lamin‐type G domain (LG) modules (α2LG1 to α2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays α2LG1‐3 and α2LG4‐5. These fragments were compared with similar modules from the laminin α1 chain and from the C–terminal region of perlecan (PGV) in several binding studies. Major heparin‐binding sites were located on the two tandem fragments and the individual α2LG1, α2LG3 and α2LG5 modules. The binding epitope on α2LG5 could be localized to a cluster of lysines by site‐directed mutagenesis. In the α1 chain, however, strong heparin binding was found on α1LG4 and not on α1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments α2LG1–3 and α2LG4‐5 also bound to fibulin‐1, fibulin‐2 and nidogen‐2 with Kd = 13–150 nM. Both tandem fragments, but not the individual modules, bound strongly to α‐dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to α‐dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell–matrix interactions and supramolecular assembly.

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.