A biological role of the carbohydrate moieties of laminin

The ways in which the carbohydrate moieties of laminin affect its cellular interactions have been examined by two different experimental approaches. In one approach, we used lectins in order to block specific carbohydrates on laminin which previously had been dried onto a plastic surface. We found that wheat germ agglutinin and Griffonia simplicifolia agglutinin I blocked the binding of the neuron-like rat pheochromocytoma cell line PC12. However, when concanavalin A was used cell binding was unaffected but neurite outgrowth was prevented, compared to controls, over a 24-h period. In the second approach we used unglycosylated laminin as a substratum on the plastic surface. We have developed a method for the purification of unglycosylated laminin from tunicamycin treated cultures of a mouse embryonal carcinoma derived cell line, M1536 B3, and have partially characterized the purified material. A mixture of unglycosylated and glycosylated laminin was selectively purified from the M1536 B3 cell lysate by an anti-EHS laminin monoclonal antibody immunoaffinity column. The unglycosylated laminin was separated from glycosylated laminin using G. simplicifolia lectin affinity chromatography. The lectins, wheat germ agglutinin, G. simplicifolia agglutinin I, and concanavalin A, did not bind to any of the subunits of unglycosylated laminin in Western blots. The unglycosylated laminin migrated as a single band in agarose-gel electrophoresis under nonreducing conditions indicating that it is a fully assembled and disulfide bonded molecule. Circular dichroism studies showed no differences between glycosylated and unglycosylated laminin, indicating similar molecular conformations. Western blots using antibodies specific for the A, B1, and B2 chains of laminin showed that unglycosylated laminin contained each of these subunits. We then performed cell binding and spreading or neurite outgrowth assays using unglycosylated laminin. A mouse melanoma cell line, B16 F1, bound to this laminin in the same numbers as to the control glycosylated laminin, but cell spreading was minimal. When this unglycosylated laminin was used as a substrate for PC12 cells neurite outgrowth was impaired; no effect was noted on the number of cells bound, compared to glycosylated laminin. We conclude from these results that once cells become bound to laminin the carbohydrate residues of that glycoprotein must be available to enable the cells to spread or to extend neurite processes.     

Functional role of carbohydrate residues in human immunoglobulin G and therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient’s serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.     

The S-type lectin from calf heart tissue binds selectively to the carbohydrate chains of laminin.

We report that the S-type lectin in calf heart tissue, termed calf heart agglutinin (CHA), binds to immobilized mouse laminin in ligand blotting and solid-phase radioligand binding assays. When compared with other glycoproteins, radioiodinated CHA binds preferentially to immobilized laminin. The binding is saturable with a Kd of 9.2 x 10(-7) M and is competitively inhibited by nonradiolabeled CHA as well as a similar lectin from porcine heart tissue. Both lactose and N-acetyllactosamine are good inhibitors of binding to laminin but binding is not inhibited by heparin. Exoglycosidase treatments demonstrated that the binding of radioiodinated CHA to laminin is not dependent on terminal sialyl-, fucosyl-, beta- or alpha-linked galactosyl residues, whereas treatment of laminin with endo-beta-galactosidase significantly decreases the lectin binding. Thus, CHA binds selectively to the poly-N-acetyllactosamine chains on complex-type Asn-linked oligosaccharides in laminin.     

Functional domains of the 67-kDa laminin receptor precursor

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.     

Functional involvement of specific carbohydrate in teratoma cell adhesion factor.

To define more precisely the telolysosome-autophagosome-autolysosome interaction during glucagon-induced autophagy in rat liver, a conceptual and mathematical model has been developed as the first step in simulation of particle behavior during the autophagic response. The present first approximation model is based on previous experimental studies, collision theory and chemical kinetics. An examination of model formulation has revealed no serious difficulties in the assumptions made and further refinement seems possible with the development of methods for quantitatively evaluating specific particle populations.     

The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans

Laminins are components of basement membranes that are required for morphogenesis, organizing cell adhesions and cell signaling. Studies have suggested that laminins function as alpha(x) beta(y) gamma(z) heterotrimers in vivo. In C. elegans, there is only one laminin beta gene, suggesting that it is required for all laminin functions. Our analysis is consistent with the role of the laminin beta as a subunit of laminin heterotrimers; the same cells express the laminin alpha, beta, and gamma subunits, the laminin beta subunit localizes to all basement membranes throughout development, and secretion of the beta subunit requires an alpha subunit. RNAi inhibition of the beta subunit gene or of the other subunit genes causes an embryonic lethality phenotype. Furthermore, a distinctive set of phenotypes is caused by both viable laminin alpha and beta partial loss-of-function mutations. These results show developmental roles for the laminin beta subunit, and they provide further genetic evidence for the importance of heterotrimer assembly in vivo.     

Evidence against a laminin receptor role for calsequestrin

Calsequestrin, a muscle calcium binding protein, has been shown to bind the extracellular matrix protein laminin and evidence has been presented that CAL (initially called aspartactin) is on the cell surface, consistent with a role as a laminin receptor (1). In this report, we present evidence that does not support a laminin receptor function for CAL. We found that CAL immunoreactivity could not be detected on live cultured chick myotubes unless they were permeabilized with detergent. Furthermore, polyclonal anti-CAL antibodies did not perturb myotube adhesion to laminin or the rate of myoblast fusion on laminin. Expression of the CAL cDNA in a melanoma cell line that was poorly adherent to laminin did not increase adhesion to laminin. In these cells, CAL could not be detected on the cell surface, and the majority of CAL was found to be secreted into the media.     

Functional contributions of carbohydrate on AIDS virus glycoprotein

Envelope glycoprotein spikes on the surface of the human immunodeficiency virus (HIV) are used by the virus to bind to cellular receptors to gain entry into target cells. As such, the envelope spikes are the targets of antibodies that can neutralize viral infectivity. Fifty percent or more of the mass of the viral-encoded surface glycoprotein of HIV, and of its close monkey relative simian immunodeficiency virus (SIV), is actually carbohydrate; it is one of the most heavily glycosylated proteins that can be found in mammals. It has been clearly demonstrated that one of the functions of this carbohydrate is to shield viral epitopes that would otherwise be the direct target of antibodies that could neutralize viral infection. In addition, it is now generally accepted that the carbohydrate on the viral envelope glycoprotein is recognized by multiple cellular lectins of the host lymphoreticular system, and these interactions play a role in the dissemination of virus within the host as well as the release of modulatory cytokines. Our work recently demonstrated fundamental differences in the composition of the carbohydrate on HIV type 1, the cause of the AIDS pandemic, versus the SIV in the sooty mangabey monkey, a natural host that does not develop disease from its infection. We now speculate that this fundamental difference in carbohydrate composition reflects evolutionary pressures on both virus and host. Furthermore, carbohydrate composition on the virus and genetic differences in carbohydrate-sensing proteins of the host could be critically important for the generalized lymphoid activation that characterizes the acquired immunodeficiency syndrome (AIDS).     

The role of laminin in cancer invasion and metastasis

Laminin, a basement membrane glycoprotein, has been implicated in a number of stages in tumour invasion and metastasis. In addition to its roles in cell adhesion and migration, laminin may be important in mediating interactions of tumour cells with the immune system and have more subtle roles in controlling metastatic behaviour. Recent work on the prognostic significance of laminin and the possible role of the molecule in antimetastasis therapy will also be discussed.     

Functional role of serotonergic neuromodulation in Aplysia.

The serotonergic metacerebral cell (MCC) of the mollusk Aplysia produces slow synaptic potentials in motor neurons of the buccal muscle, and increases the rate of ongoing rhythmic burst output of the buccal ganglion. In addition, the MCC acts peripherally to enhance the strength of buccal muscle contractions that are produced by firing of motor neurons. The potentiation of contraction is not associated with any detectable changes of resting membrane potential of muscle cells. Although MCC activity produces a small enhancement of excitatory junctional potentials, several experiments clearly indicate that the MCC has a direct potentiating effect on excitation-contraction coupling. The data suggest that potentiation of contraction might be mediated by cAMP. For example, activity of the MCC enchances the rate of accumulation of cAMP in buccal muscle, application of phosphodiesterase resistant analogs of cAMP potentiates muscle contraction, and a phosphodiesterase inhibitor enhances the effect of MCC stimulation. Recordings from free-moving animals indicate that the MCC becomes activated by exposure of the animal to food stimuli, and that the activation parallels the presence of a food-arousal state. Food-arousal is characterized by enhanced strength and increased frequency of biting responses. Both these effects can result from activity of the MCC. Thus, in this system, modulatory synaptic actions function to provide the substrate for a type behavioral modulation.     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.     

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.     

The role of carbohydrate in the glycoenzyme invertase of Neurospora crassa.

Data obtained concerning the carbohydrate moieties of the glycoenzyme invertase (EC 3.2.1.26, beta-D-fructofuranoside fructohydrolase) from Neurospora crassa were consistent with a linkage of some carbohydrate chains by O-glycosidic bonds to serine and threonine residues; the possibility of N-glycosylamine linkage of some of the carbohydrate to the amide group of asparagine is also indicated. The invertase was remarkably stable on storage at low temperatures. Oxidation of the carbohydrate residues in the enzyme by sodium periodate markedly affected the heat-stability of the enzyme. It is suggested that the carbohydrate moieties function as stabilizers of the tertiary structure of the glycoenzyme.     

The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons

Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel role for macrophage: antigen discrimination of distinct carbohydrate bonds.

Velocity sedimentation-derived subpopulations of peritoneal exudate macrophages have been pulsed with a variety of TNP-coupled carbohydrate/protein antigens and investigated for their ability to present those antigens in immunogenic form to whole spleen cell populations. The data presented indicate that while no difference is seen in the ability of the various subpopulations examined to present protein antigens for an antibody response, different cells are certainly involved in the antigen handling of carbohydrate antigens for an antibody response. Moreover, it seems that individual macrophage subpopulations can only interact with certain subpopulations of B lymphocytes to induce antibody responses to the same immunogenic determinants.     

On the role of the collagen carbohydrate residues in the platelet. Collagen interaction.

It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To test this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantly reduced its ability to interact with platelets. The extent of inhibition paralleled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.     

The role of laminin in embryonic cell polarization and tissue organization

Genetic analyses have revealed that members of the laminin glycoprotein family are required for basement membrane assembly and cell polarization, with subsequent effects on cell survival and tissue organization during metazoan embryogenesis. These functions depend upon the cooperation between laminin polymerization and cell anchorage mediated via interactions with beta1-integrins, dystroglycan, and other cell surface receptors.