Y Yamada

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

A major nidogen binding site of mouse laminin was previously localized to about three EGF‐like repeats (Nos 3–5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3‐5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3‐5 and nidogen could be efficiently converted into a covalent complex by cross‐linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3‐5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3‐5 was indicated from its protease resistance and the complete loss of cross‐reacting epitopes as well as of nidogen‐binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF‐like repeats 3–4 and 4–5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen‐binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28‐residue signal peptide. The data support a previously proposed dumb‐bell model of nidogen by demonstrating a large N‐terminal globular domain (641 residues), five EGF‐like repeats constituting the rod‐like domain (248 residues) and a smaller C‐terminal globule (328 residues). Two more EGF‐like repeats interrupt the N‐terminal and terminate the C‐terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta‐hydroxylation, two N‐linked carbohydrate acceptor sites and, within one of the EGF‐like repeats an Arg‐Gly‐Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C‐terminal globule but not yet precisely localized.

Laminin-1 and Laminin-2 G-domain Synthetic Peptides Bind Syndecan-1 and Are Involved in Acinar Formation of a Human Submandibular Gland Cell Line

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin α1 and α2 chains. The α1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous α2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-β1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-β1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both β1-integrins and syndecan-1.

Laminin A chain synthetic peptide which supports neurite outgrowth

Neurons from peripheral and central nervous tissue as well as from established cell lines respond to low concentrations of laminin with rapid extension of axon-like processes. Two sites on laminin have been identified which stimulate neurite outgrowth, the major site residing at the end of the long arm of laminin. Recently laminin has been cloned and sequenced allowing for synthetic peptides to be prepared and tested for biological activity. We report here that antisera against synthetic peptides corresponding to A and to B1 chain sequences at the end of the long arm can partially inhibit laminin-mediated neurite outgrowth. Further, we show that a 19 amino acid synthetic peptide (CSRARKQAASIKVAVSADR) from the long arm of the laminin A chain is capable of stimulating neuronal-like process formation to almost the same extent as laminin and competes with laminin for stimulation of neurite outgrowth.

Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening

Laminin-1, a major component of basement membranes, consists of three different chains designated α1, β1, and γ1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin γ1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the γ1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-α2β1integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin γ2 chain. These results suggest that the specific sequences on the laminin γ1 chain are biologically active and interact with distinct cell surface receptors.

Laminin‐like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

The basement membrane glycoprotein laminin‐1 is a potent stimulator of neurite outgrowth. Although a variety of laminin isoforms have been described in recent years, the role of alternative laminin isoforms in neural development remains largely uncharacterized. We found that a polyclonal antibody raised against the α1, β1, and γ1 chains of laminin‐1 and a monoclonal antibody raised against the α2 chain of laminin‐2 detect immunoreactive material in neuronal cell bodies in the developing mouse cerebellum. In addition, laminin‐1‐like immunoreactivity was found in cell types throughout the cerebellum, but laminin‐α2‐like immunoreactivity was restricted to the Purkinje cells. Purified laminin‐1 and laminin‐2 stimulated neurite outgrowth in primary cultures of mouse cerebellar granule neurons to a similar extent, whereas the synthetic peptides tested appeared to be active only for cell adhesion and not for stimulation of neurite outgrowth. The E8 proteolytic fragment of laminin‐1 contained full neurite outgrowth activity. The identity of laminins expressed in granule neurons was also examined by Western blotting; laminin‐like complexes were associated with the cell and appeared to have novel compositions. These results suggest that laminin‐like complexes play important roles in cerebellar development. J. Neurosci. Res. 54:233–247, 1998.

Structure and Biology of the Laminin-Nidogen Complex

A large variety of cells in the body are exposed to extracellular matrices which affect cell function and behavior. Basement membranes are prominent matrix structures and have been shown to consist of several collagenous and non-collagenous glycoproteins and proteoglycans (Martin and Timpl, 1987). Laminin is one of the major and ubiquitous components that was originally isolated and characterized from a tumor basement membrane as a cross-shaped, multidomain protein (Timpl et al., 1979; Engel et al., 1981). It is considered to be one of the important cell-binding proteins present in basement membranes and in addition possesses binding potentials for other matrix components. Another non-collagenous glycoprotein, nidogen/entactin, was initially discovered as a sulfated 158kD polypeptide (Carlin et al., 1981) and shown to have a characteristic dumb-bell structure (Paulsson et al., 1986; 1987). Even though nidogen is also an ubiquitous constituent of basement membranes its function is not immediately apparent. However, several pieces of indirect evidence from cell culture and tissue studies (Dziadek and Timpl, 1985) provided initial clues about the possible role of nidogen, by showing that it forms tight, stoichiometric, noncovalent complexes with laminin in situ.