Mouse Laminin Research Articles

Active sites in the carboxyl-terminal region of the laminin alpha chain in Drosophila neuronal cell spreading.

An established Drosophila neuronal cell line (BG2-c6) proved to be useful to analyze laminin-mediated cell spreading and signal transduction [Takagi et al. (2000) Biochem Biophys Res Commun 270:482-487]. Here, we report, in addition to the whole molecule, the truncated alpha chain of Drosophila laminin (containing the entire carboxyl-terminal globular domain) and two dodecapeptides corresponding to the cell-binding sites identified in the alpha1 chain of mouse laminin were also active to stimulate BG2-c6 cell spreading. Our previous study [Takagi et al. (1996) J Biol Chem 271:18074-18081] revealed that these recombinant protein and synthetic peptides promoted neurite outgrowth in the primary cell culture system prepared from Drosophila embryo. Therefore, the similar effects by these proteins and peptides suggest the presence of a common mechanism of laminin and neuronal cell interaction working in both primary and established cells. One of the two active peptides contains the sequence SIKVGV. Its murine counterpart carries the sequence SIKVAV by which the interaction of laminin and cells is mediated. Furthermore, laminin-dependent BG2-c6 cell spreading was inhibited by heparin. This observation suggests that cell surface glycoproteins participate in the interaction of laminin and BG2-c6 cells.

Changes in pools of autoantibodies and anti-bacterial antibodies in patients suffering from recurrent infections of the urinary tract and undergoing bacterial immunization treatment

Antibodies (Abs) (IgM, IgA, IgG and IgG subclasses) specific for several uropathogenic strains ( Escherichia coli, Pseudomonas sp. and Klebsiella sp.) as well as anti-phospholipids, anti-β 2-glycoprotein I and anti-laminin antibodies were analyzed in the sera of 20 patients with long-lasting uncomplicated recurrent infections of the lower urinary tract who underwent immunization treatment with a mixture of heat-inactivated bacteria. Immunization had a dual effect: a marked prolongation of the infection-free period in more than half of tested patients (which could be related to the profiles of anti-bacterial antibodies), and the induction of a significant decrease in autoreactivity. The results obtained showed that prolonged infections resulted in a significant rise in IgG specific for phospholipids, β 2-glycoprotein I and mouse laminin. However, irrespective of the effect on urinary tract infection per se, immunization induced a noticeable decrease in reactivity toward those antigens (Ag). The most abundant autoantibodies prior to immunization treatment were of IgG2 subclass. A statistically significant decrease in phospholipid specific antibodies belonging to this subclass, and in the concentration of Y7 cross-reactive idiotope, registered only in the responder group; this indicates the significance of natural antibody pool involvement in a proper anti-bacterial immune response.

Changes in pools of autoantibodies and anti-bacterial antibodies in patients suffering from recurrent infections of the urinary tract and undergoing bacterial immunization treatment

Antibodies (Abs) (IgM, IgA, IgG and IgG subclasses) specific for several uropathogenic strains (Escherichia coli, Pseudomonas sp. and Klebsiella sp.) as well as anti-phospholipids, anti-β2-glycoprotein I and anti-laminin antibodies were analyzed in the sera of 20 patients with long-lasting uncomplicated recurrent infections of the lower urinary tract who underwent immunization treatment with a mixture of heat-inactivated bacteria. Immunization had a dual effect: a marked prolongation of the infection-free period in more than half of tested patients (which could be related to the profiles of anti-bacterial antibodies), and the induction of a significant decrease in autoreactivity. The results obtained showed that prolonged infections resulted in a significant rise in IgG specific for phospholipids, β2-glycoprotein I and mouse laminin. However, irrespective of the effect on urinary tract infection per se, immunization induced a noticeable decrease in reactivity toward those antigens (Ag). The most abundant autoantibodies prior to immunization treatment were of IgG2 subclass. A statistically significant decrease in phospholipid specific antibodies belonging to this subclass, and in the concentration of Y7 cross-reactive idiotope, registered only in the responder group; this indicates the significance of natural antibody pool involvement in a proper anti-bacterial immune response.

Functional idiotypic mimicry of an adhesion- and differentiation-promoting site on acetylcholinesterase.

Acetylcholinesterase mediates cell adhesion and neurite outgrowth through a site associated with the peripheral anionic site (PAS). Monoclonal antibodies raised to this site block cell adhesion. We have raised anti-idiotypic antibodies to one of these antibodies. The anti-idiotypic antibodies recognized the immunogenic antibody and non-specific mouse IgG, but not acetylcholinesterase. Five antibodies (out of 143 clones, an incidence of 3.5%) were able to promote neurite outgrowth in human neuroblastoma cells in vitro in a similar manner to acetylcholinesterase itself, suggesting that these antibodies carry an internal image of the neuritogenic site. Two of the antibodies were significantly more effective (P < 0.01) than acetylcholinesterase in this regard. The antibodies also bound specifically to mouse laminin-1 and human collagen IV, as does acetylcholinesterase. This binding was displaced by unlabelled antibody, as well as by acetylcholinesterase itself, indicating competition with acetylcholinesterase. We have also investigated the development of anti-anti-idiotypic antibodies in mice in vivo, and have observed that four of these (out of 318 clones, an incidence of 1.26%) mimic the idiotypic antibody and abrogate adhesion in neuroblastoma cells. We have thus demonstrated functional mimicry of the neuritogenic site on acetylcholinesterase in anti-idiotypic antibodies, enhancement of this activity in one antibody, and mimicry of the idiotypic antibody site in anti-anti-idiotypic antibodies. Implications of these findings for differentiation-promoting cancer therapy are discussed.

Expression of Y7 Cross‐Reactive Idiotope on Human IgM Molecules

In this paper we report data regarding the IgM Y7 cross‐reactive idiotope (CRIo) obtained by analysis of: 1) its V‐gene subgroup dependance, 2) the frequency of its expression on human monoclonal IgMs and IgM molecules from normal and pathological sera. Furthermore, comparison of epitopic repertoire and nature of binding of human monoclonal IgMs expressing Y7 CRIo was performed to confirm the natural antibody properties of these molecules. IgM isolated from sera of patient DJ (IgM DJ) which expresses the Y7 idiotope has been classified to VH3/VL2 subgroup. From ten IgMs tested only IgM from patient RD (IgM RD) has been shown to express Y7 idiotope. Y7+ human IgMs bound to ssDNA, lactic acid bacteria, mouse laminin, porcine thyroglobulin and mouse IgG. Higher percentage of the expression of Y7 CRIo was detected in the sera of patients suffering from autoimmune diseases such as lupus, rheumatoid arthritis and psoriasis vulgaris as well as in patients suffering from chronic infections of the lower urinary tract. Antigen binding repertoire and properties of Y7+ monoclonal IgM, frequency of Y7 expression on monoclonal IgMs and its concentration in normal and pathological sera indicate the important biological role of this CRIo within the immune system.

Multifunctional peptide fibrils for biomedical materials.

The Ile-Lys-Val-Ala-Val (IKVAV) containing peptide, A208 (AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108), was recently found to form amyloid-like fibrils. Fibril formation is critical for its biological activities, including promotion of cell adhesion and neurite outgrowth. In the present study, we designed multifunctional peptide fibrils using the A208 peptide and an Arg-Gly-Asp (RGD)-containing fibronectin active sequence for biomedical applications. The fibronectin active sequence GRGDS (FN) or a scrambled sequence RSGGD (SC) were conjugated to either A208 or to A208S (AASVVIAKSADR), a scrambled peptide of A208, with a glycine as a spacer. The FN-A208 and SC-A208 peptides formed a gel and were stained with Congo red similar to that of A208, but FN-A208S and SC-A208S did not form a gel. These results indicate that FN-A208 and SC-A208 form amyloid-like fibrils similar to A208. A208 and SC-A208 promoted cell attachment with filopodia formation, and this adhesion was inhibited by the IKVAV-containing peptide, but not by EDTA or a GRGDS peptide. FN-A208 promoted cell attachment with well-organized actin stress fibers, and this adhesion was partially inhibited by either EDTA, GRGDS, or IKVAV. These data suggest that A208 binds to only IKVAV receptor(s) while the FN-A208 interacts with both integrins and the IKVAV receptor(s). We conclude that multifunctional peptide fibrils can be designed by conjugation of active peptides on A208 and that this construct has potential to serve as a bioadhesive for tissue regeneration and engineering.

Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

Objectives: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic αgalactose (Galα) antigens in the doner tissue. Design and methods: We have measured the binding potentials of Galα detecting lectins and antibodies, including a naturally occurring subfraction from human serum, to Galα containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates. Results: Galα reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin and neoglycoproteins were treated with α-galactosidase and subsequently incubated with antibodies and lectins. The enzyme treatment was more deleterious on antibody binding than on lectin binding. Conclusion: Antibodies and lectins may bind to different galactose determinants on the glycoproteins. Two anti-Galα1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galα-antigens with varying specificities. Binding results obtained after digestion with α-galactosidase indicate that some xenoreactive Galα groups are not directly accessible for removal by the enzyme.

Immunization of naïve mice with mouse laminin-1 affected pregnancy outcome in a mouse model.

Laminins have important roles during placental and embryonic development. The aim of our study was to determine if active immunization of mice with laminin-1 could elicit an autoimmune response, and induce features of reproductive failure. BALB/c mice were immunized with mouse laminin-1. Autoantibodies to laminin-1 were measured by enzyme-linked immunosorbent assay. Pregnant mice were killed on day 14 of pregnancy and examined for pregnancy outcome. Mice immunized with laminin-1 developed elevated levels of anti-laminin-1 auto-antibodies contrary to the control group. A higher fetal resorption rate was found in the laminin-1 immunized group (23.8%) compared with that of the control group (12.2%), and was even higher in the subgroup of those animals with very high levels of anti-laminin-1 (P < 0.01). Laminin-1 immunized mice also had lower fetal and placental weights. Active immunization with laminin-1 followed by elevated circulating anti-laminin-1 antibodies results in reproductive failure manifested by a higher fetal resorption rate.

Identification of an Upstream Enhancer in the Mouse Lamininα1 Gene Defining Its High Level of Expression in Parietal Endoderm Cells

Laminin-1 is the major component of the embryonic basement membrane and consists of alpha1, beta1, and gamma1 chains. The expression of laminin-1 is induced in mouse F9 embryonal carcinoma cells upon differentiation into parietal endoderm through transcriptional up-regulation of the genes encoding these subunits. Here, we identified a 435-bp enhancer in the 5'-flanking region of the mouse laminin alpha1 (LAMA1) gene that activated its transcription in a differentiation-dependent manner. This enhancer was also active in PYS-2 parietal yolk sac-derived cells but not in NIH/3T3 fibroblasts, indicating that it was a parietal endoderm-specific enhancer. This enhancer was also active in Engelbreth-Holm-Swarm (EHS) tumor-derived cells characterized by excessive production of laminin-1 and other basement membrane components, suggesting that EHS tumors have a transcriptional control mechanism similar to that of parietal endoderm cells. Electrophoretic mobility shift analyses revealed four protein binding sites (PBS1-PBS4) in the 435-bp region. However, these DNA-binding proteins were detected not only in parietal endoderm cells (i.e. differentiated F9 cells, PYS-2 cells, and EHS tumor-derived cells) but also in undifferentiated F9 cells and NIH/3T3 cells. Mutational analyses revealed that three of these binding sites (PBS2, PBS3, and PBS4) function synergistically to confer the parietal endoderm-specific enhancer activity. The proteins binding to PBS2 and PBS4 were identified as the Sp1/Sp3 family of transcription factors and YY1, respectively.

Spontaneous muscular dystrophy caused by a retrotransposal insertion in the mouse laminin α2 chain gene

We identified a novel spontaneous mouse model of human congenital muscular dystrophy with laminin α2 chain deficiency, named dy Pas /dy Pas . Homozygous animals rapidly developed a progressive muscular dystrophy leading to premature death. Immunohistological and biochemical analyses demonstrated the absence of laminin α2 chain expression in skeletal muscle. Analysis of the laminin α2 chain cDNA showed the insertion of the long terminal repeat of an intracisternal A-particle gene. In addition, a 6.1 kb insertion composed of retrotransposon elements was identified in the Lama2 sequence. The dy Pas /dy Pas mouse is thus the first spontaneous mutant with a complete laminin α2 chain deficiency in which the mutation has been identified.

Human acetylcholinesterase binds to mouse laminin-1 and human collagen IV by an electrostatic mechanism at the peripheral anionic site

Acetylcholinesterase (EC 3.1.1.7; AChE) is known to induce neurite outgrowth and differentiation, but its ligands are as yet unknown. Laminin-1 and collagen IV were investigated as potential ligands for AChE. We observed specific saturable binding of biotinylated human AChE to mouse laminin and human collagen, with K d values of 4.9482 nM (SE 0.3145 nM) and 1.1617 nM (SE 0.1921 nM) respectively. Peripheral anionic site inhibitors (fasciculin, BW284c51, propidium and gallamine) also significantly reduced binding with fasciculin being the most effective. Significant reductions in AChE–laminin and AChE–collagen interactions were produced by a monoclonal anti-AChE antibody known to react with the peripheral anionic site, and a partial reduction with an antibody that partially recognises the site. Self-association of AChE was also observed ( K d =16.3235 nM; SE 5.8120 nM); increasing markedly at low pH, but not significantly affected by either inhibitors or antibodies, suggesting a non-specific aggregation phenomenon. Binding to laminin and collagen was significantly reduced by increasing ionic strength and decreasing pH, indicating a dominant role for electrostatic interactions, and suggesting that the site may be different from the hydrophobic site identified for the AChE–amyloid interaction.

Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain

Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin α1 chain peptides form amyloid-like fibrils

The Ile-Lys-Val-Ala-Val (IKVAV) sequence derived from laminin-1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV-containing peptide (LAM-L: AASIKVAVSADR, mouse laminin α1 chain 2097–2108). The LAM-L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross-β X-ray diffraction pattern. Further, infrared spectra of LAM-L suggested a β-sheet structure. These results indicate that LAM-L forms amyloid-like fibrils. We also examined amyloid-like fibril formation of LAM-L analogs. The neurite outgrowth activity of the LAM-L analogs was closely related to their amyloid-like fibril formation.

Binding of Mouse Nidogen-2 to Basement Membrane Components and Cells and Its Expression in Embryonic and Adult Tissues Suggest Complementary Functions of the Two Nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin γ1 chain (γ1III4) is important for kidney development and for nerve guidance. Other proteins may also bind γ1III4, although human nidogen-2 binds poorly to the mouse laminin γ1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and γ1III3-5, which both contain the γ1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial–mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin γ1 chain is dependent on epithelial–mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by α3β1 and α6β1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.

Tissue-dependent expression of matrix proteins in human endothelin-1 transgenic mice.

Endothelin-1 (ET-1) is a potent pro-fibrotic growth factor. However, little is known about its specific effects on the synthesis of matrix proteins in vivo. We used male 12-month-old ET-1 transgenic mice characterized by transgene expression in the kidney and (to a lesser extent) in the heart. Global cardiac and renal matrix protein synthesis was analysed after Sirius Red and periodate-Schiff staining. Specific expression of collagen types I, III and IV, laminin and fibronectin was examined using immunohistochemistry followed by computer-aided image analysis. Analysis of blood pressure revealed that mean arterial blood pressure was similar in ET-1 transgenic mice and controls. The total cardiac matrix protein content was increased in the myocardium of ET-1 transgenic mice. Analysis of specific cardiac matrix proteins showed increased cardiac expression of collagen type III (+211%; P<0.001) and laminin (+128%; P<0.01) in transgenic mice. The expression of collagen types I and IV and fibronectin was not altered. Global analysis of renal matrix proteins confirmed earlier studies showing pronounced interstitial fibrosis and glomerulosclerosis. Laminin expression was markedly increased in the glomerula (+152%; P<0.01) and even more so in the interstitium (+211%; P<0.001), whereas expression of collagen type III was reduced in glomerula (-48%; P<0.01) and interstitial tissue (-55%; P<0.01) of ET-1 transgenic mice. In conclusion, a primary overexpression of ET-1 does not cause uniformly enhanced synthesis of matrix proteins. In contrast, the effects of ET-1 on the matrix protein pattern is tissue-specific. The major renal and cardiac alterations in matrix proteins induced by ET-1 is a marked enhancement of laminin expression.

The synthesis of deoxy-α-Gal epitope derivatives for the evaluation of an anti-α-Gal antibody binding

α-Gal epitopes (also termed as α-Gal) are carbohydrate structures bearing the α- d-Gal-(1→3)-β- d-Gal terminus 1 and are known to be the antigen responsible for antibody-mediated hyperacute rejection in xenotransplantation. Terminal 2-, 3-, 4-, and 6-deoxy-Gal derivatives of α-Gal were synthesized. Inhibition ELISA using mouse laminin was established to determine the binding affinity of the synthesized α-Gal derivatives. 4-Deoxy-α-Gal derivative 7 showed a significant reduction in antibody recognition. The IC 50 value was 15-fold poorer than the standard α-Gal epitopes α- d-Gal-(1→3)-β- d-Gal-(1→4)-β- d-Glc-NHAc ( 39) and α- d-Gal-(1→3)-β- d-Gal-(1→4)-β- d-Glc-OBn ( 40). A similar observation was seen with 2-deoxy-α-Gal derivative 5, whose IC 50 value was nearly tenfold higher than the standards. Interestingly, substitution at the terminal 3-position resulted in only a fourfold decrease in antibody recognition, suggesting a possible point of future derivation. Finally, 6-deoxy-α-Gal derivative 8 exhibited similar antibody recognition to both α-Gal epitope 39 and α-Gal epitope 40. This strongly suggests that derivatization at the 6-position can be accomplished without loss of antibody recognition. These findings can be utilized for the future design of other α-Gal derivatives.

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins.

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.

Laminin α5 Is Required for Lobar Septation and Visceral Pleural Basement Membrane Formation in the Developing Mouse Lung

Laminin α/β/γ heterotrimers are the major noncollagenous components of all basement membranes. To date, five α, three β, and three γ chains have been identified. Laminin α5 is expressed early in lung development and colocalizes with laminin α1. While laminin α1 expression in the lung is restricted to the embryonic period, laminin α5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin α5 gene Lama5 causes embryonic lethality at E14–E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5−/− mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin α4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin α5, which was found to be the only laminin α chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.

Production of human monoclonal antibodies against Galalpha(1-3)Gal.

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galalpha(1-3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human-mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.

Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin α3B and α5 chains

The N-terminal sequences of mouse laminin α3B and α5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45–65kDa) from this region yielding properly folded proteins, which were secreted from HEK-293—EBNA cells. Pepsin digestion of these fragments yielded products of 25–35kDa, which consisted only of domain V. The αVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the α3B and α5 chains being more active than those from α1 and α2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (Kd = 0.02 to &amp;gt; 6μM) for the binding to laminin-1. This indicated that laminins containing α3B or α5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for α1/α2, moderate for α3B, whereas no binding was observed for α5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against α3BVI/V and α5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of α3B, β3 and γ2 chains.