Assessing the severity of the small inframe deletion mutation in the alpha-subunit of beta-hexosaminidase A found in the Turkish population by reproducing it in the more stable beta-subunit.

GM(2) gangliosidoses are a group of panethnic lysosomal storage diseases in which GM(2) ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the alpha- or beta-subunits of beta- N -acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the alpha-subunit of Hex has been found in five Turkish patients with infantile Tay-Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive alpha-protein in its precursor form. Because the alpha- and beta-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the beta-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the alpha homodimer. Thus, mutations that completely destabilize the alpha-subunit can often be partially rescued if expressed in the aligned positions in the beta-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267-1278) in the beta-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the beta-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable beta-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.

Cloning and molecular characterization of two splice variants of a new putative member of the Siglec-3-like subgroup of Siglecs.

The sialic acid binding immunoglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within this Siglec family there exists a subgroup of molecules which bear a very high degree of homology with the molecule Siglec-3 (CD33), and has thus been designated the Siglec-3-like subgroup of Siglecs. The members of this subgroup have been localized to chromosome 19q13.4, through both in situ hybridization and precise genomic mapping at the nucleotide level. Through the positional cloning approach we have identified and characterized a Siglec-like gene (SLG), a putative novel member of the Siglec-3-like subgroup of Siglecs. We have characterized the complete genomic structure of SLG, as well as two alternative splice variants, and determined its chromosomal localization. The short isoform, SLG-S, consists of seven exons, with six intervening introns, while the longer isoform, SLG-L, consists of eight exons and seven intervening introns. The SLG gene is localized 32.9 kb downstream of Siglec-8 on chromosome 19q13.4. The putative SLG-S and SLG-L proteins, of 477 and 595 amino acid residues, respectively, show extensive homology to many members of the Siglec-3-like subgroup. This high degree of homology is conserved in the extracellular Ig-like domains, as well as in the cytoplasmic tyrosine-based motifs. Interestingly, the SLG-L protein contains two N-terminal V-set Ig-like domains, as opposed to SLG-S and other Siglec-3-like subgroup members which contain only one such domain. Through RT-PCR we have examined the expression profile of both SLG splice variants in a panel of human tissues and have found that SLG-S is highly expressed in spleen, small intestine and adrenal gland, while SLG-L exhibits high levels of expression in spleen, small intestine, and bone marrow. This gene is quite likely the latest novel member of the CD33-like subgroup of Siglecs, and given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec7.

Myelin-associated glycoprotein binding to gangliosides. Structural specificity and functional implications.

Myelin-associated glycoprotein (MAG), which mediates certain myelin-neuron cell-cell interactions, is a lectin that binds to sialylated glycoconjugates. Gangliosides, the most abundant sialylated glycoconjugates in the brain, may be the functional neuronal ligands for MAG. Cells engineered to express MAG on their surface adhered specifically to gangliosides bearing an alpha 2,3-linked N-acetylneuraminic acid on a terminal galactose, with the following relative potency: GQ1b alpha >> GD1a, GT1b >> GM3, GM4 (GM1, GD1b, GD3, and GQ1b did not support adhesion). MAG binding was abrogated by modification of the carboxylic acid, any hydroxyl, or the N-acetyl group of the ganglioside's N-acetylneuraminic acid moiety. Related immunoglobulin (Ig) superfamily members either failed to bind gangliosides (CD22) or bound with less stringent specificity (sialoadhesin), whereas a modified form of MAG (bearing three of its five extra-cellular Ig-like domains) bound only GQ1b alpha. Enzymatic removal of sialic acids from the surface of intact nerve cells altered their functional interaction with myelin. These data are consistent with a role for gangliosides in MAG-neuron interactions.

Binding specificities of the sialoadhesin family of I-type lectins. Sialic acid linkage and substructure requirements for binding of myelin-associated glycoprotein, Schwann cell myelin protein, and sialoadhesin.

The carbohydrate binding specificities of three sialoadhesins, a subgroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1balpha >> GD1a = GT1b >> GM3 = GM4 >> GM1, GD1b, GD3, GQ1b (nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal alpha2,3- or alpha2,8-linked sialic acids with the following rank-order potency of binding: GQ1balpha > GD1a = GD1b = GT1b = GM3 = GM4 > GD3 = GQ1b >> GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid acetamido group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.

Regulation of myelin-associated glycoprotein binding by sialylated cis-ligands.

Myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP) are highly glycosylated members of a newly defined family of cell adhesion molecules belonging to the immunoglobulin superfamily that recognize terminal sialic acid residues on N- and O-linked oligosaccharides. The importance of the N-linked oligosaccharides on MAG were determined by removal of the eight predicted carbohydrate addition sites by site-directed mutagenesis. The results suggest that all eight N-linked glycosylation sites are utilized in COS cells. N-linked glycosylation does not appear to be required for sialic acid-dependent MAG binding to erythrocytes. However, N-linked glycosylation of MAG does play a role in the proper folding of MAG. It was also shown that sialylation in the host cell expressing MAG and SMP could inhibit binding to erythrocytes. The degree to which SMP and MAG erythrocyte binding was affected by sialylation in the host cell was dependent on (a) the level at which MAG was expressed on the surface of the host cell and (b) the presence of MAG ligands on the host cell. The data suggest that cis-ligands on the host cell compete with trans-ligands on the target cell for the binding site(s) on MAG.

Cytotoxicity of B72.3XOKT3 bispecific antibody recognizing human colon cancer.

Bispecific monoclonal antibodies can be used to redirect peripheral blood lymphocytes against tumor cells. In the present study, a murine bispecific monoclonal antibody was developed using somatic hybrydization. The antibody has two different binding arms: one arm directed against human CD3 receptor expressed on T-lymphocytes and the other against tumor associated glycoprotein TAG-72, expressed on human carcinomas, such as colon, breast, and pancreas. Partially purified antibody was capable of inducing human T-cell proliferation and preventing growth of colon cancer cell line in nu/nu mice in a tumor neutralization assay.

Conservation of functionally important epitopes on myelin associated glycoprotein (MAG).

Phylogenetic conservation of protein domains often points to functionally important regions. As a step toward mapping these sites on myelin associated glycoprotein (MAG) we have determined the species distribution of epitopes recognized by a panel of anti-MAG antibodies (Ab). Monoclonal antibodies (mAb) B11F7, GenS3 and 28 recognized MAG only in mammalian species. However, the mAb 513 which inhibits MAG binding recognized a conformational epitope in a wider distribution of species including, human (Homo sapiens), bovine (Bos taurus), rat (Rattus norvegicus), chicken (Gallus gallus), quail (Coturnix coturnix japonica), lizard (Iguana iguana), snake (Thamnophis sirtalis), frog (Xenopus laevis) and turtle (all tetrapods) but not in goldfish (Crassius aurata) (a teleost). However, only MAG from mammals was shown to bind rat dorsal ganglion neurons (DRGs) suggesting that structures additional to those recognized by mAb 513 must be involved in function. Antibody 28, on the other hand, recognized only MAG species which bound to neurons, suggesting that this epitope, in comparison with mAb 513, more closely represented the functionally important region of MAG. Observed species differences in glycosylation of MAG may be functionally significant. A newly developed polyclonal Ab against MAG recognized the protein in tetrapods and teleosts, but not chondricthyes. The results show that MAG is present in a wide spectrum of species.

Functional topography of myelin-associated glycoprotein. II. Mapping of domains on molecular fragments.

The myelin-associated glycoprotein (MAG), an adhesion molecule of the immunoglobulin (Ig) superfamily with five Ig-like domains, was investigated with regard to its binding site(s) for the neuronal cell surface, collagen I, and heparin, using a panel of new monoclonal antibodies and cyanogen bromide cleavage fragments of MAG. All antibodies generated competed with each other for binding to MAG, indicating that they reacted with identical or closely related epitopes. Mapping of the reactive epitopes on recombinant deletion fragments of MAG expressed by Chinese hamster ovary (CHO) fibroblasts showed reactivity of monoclonal antibody 513 with domains I, II, and III, comprising the amino-terminal end of the extracellular domain. Monoclonal antibody 15 recognized domain III only. Binding of MAG-containing liposomes to neurons was blocked by antibodies 15 and 513. Cyanogen bromide (CNBr) fragments of domains I, II, and III bound to collagen type I under isotonic buffer conditions. CNBr fragments containing domains I and II were involved in binding to heparin. These observations suggest that domain III may be important for binding to the neuronal cell surface receptor for MAG, while domains I, II, and III interact with collagen type I and domains II and III with heparin.

Myelination in the absence of myelin-associated glycoprotein.

The hypothesis that myelin-associated glycoprotein (MAG) initiates myelin formation is based in part on observations that MAG has an adhesive role in interactions between oligodendrocytes and neurons. Furthermore, the over- or underexpression of MAG in transfected Schwann cells in vitro leads to accelerated myelination or hypomyelination, respectively. Here we test this idea by creating a null mutation in the mag locus and deriving mice that are totally deficient in MAG expression at the RNA and protein level. In adult mutant animals the degree of myelination and its compaction are normal, whereas the organization of the periaxonal region is partially impaired. Mutant animals show a subtle intention tremor. Our findings do not support the widely held view that MAG is critical for myelin formation but rather indicate that MAG is necessary for maintenance of the cytoplasmic collar and periaxonal space of myelinated fibres.

High-resolution mapping of GenS3 and B11F7 epitopes on myelin-associated glycoprotein by expression PCR.

The GenS3 and B11F7 monoclonal antibodies (MAbs) have been widely used for biochemical and immunocytochemical experiments on myelin-associated glycoprotein (MAG), a cell adhesion molecule mediating the interaction between myelinating glia and axons. We have mapped the epitopes to within several amino acids on Ig domain 2 (D2) (amino acids 167-77) and domain 4 (D4) (amino acids 375-388) for GenS3 and B11F7, respectively. Domain deletion and substitution mutants of the MAG cDNA were first used to map the epitopes to a given domain. In the cases of GenS3, insertion mutants were used to resolve the epitope to a small region of D2. For the B11F7 epitope, a novel technique combining PCR and in vitro transcription and translation was used to generate small C-terminal deletions and map the epitope to 13 amino acids. Then, inhibition by peptides corresponding to the GenS3 (ELRPELSWLGHE; amino acids 167-177) and B11F7 (QLELPAVTPEDDGE; amino acids 375-388) epitopes was used to confirm the position of the epitopes based on the mutant data. Interestingly, the GenS3 epitope maps to a region predicted to be sequestered within the hydrophobic core of D2. This is consistent with the inability of GenS3 to recognize the epitope in native MAG; GenS3 epitope recognition occurs only in denatured MAG, where the epitope is more accessible. With the definition of the GenS3 and B11F7 epitopes, these antibodies will be useful for further structure-function studies on MAG.

Molecular characterization of the Schwann cell myelin protein, SMP: structural similarities within the immunoglobulin superfamily.

The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAG) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAG, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily.

Recombinant myelin-associated glycoprotein confers neural adhesion and neurite outgrowth function.

Myelin-associated glycoprotein (MAG) cDNA clones for the small (p67) and large (p72) forms were expressed in heterologous cells. Purified recombinant MAG protein was incorporated into fluorescent liposomes, and both forms were shown to bind predominantly to neurites in DRG or spinal cord cultures. This adhesion was completely blocked by Fab fragments of monoclonal anti-MAG antibody. Liposomes prepared with the control protein glycophorin or no protein failed to bind neurites. Small cerebellar neurons, which are not myelinated in vivo, failed to bind MAG liposomes. In a second test of function, p67 MAG-transfected fibroblasts were markedly enhanced in their ability to promote DRG neurite extension over a 2 day culture period compared with control fibroblasts not expressing MAG. Neurite extension was blocked by anti-MAG antibodies. These results show that both forms of MAG can facilitate the interactions between glial cells and neurites that ultimately lead to myelin formation.

Differential splicing of MAG transcripts during CNS and PNS development.

Myelin-associated glycoprotein (MAG) is expressed on the surface of glial cells and is thought to act as a glial-neuronal adhesion molecule during early stages in the myelination process. Sequencing of several cDNA clones predicted the existence of two classes of MAG mRNAs which differ in the presence or absence of a 45 nucleotide insert near the 3' end. These two mRNAs are sufficient to encode the two MAG proteins previously described (p67MAG and p72MAG) and show that they differ only in their carboxyl terminal regions. The results of RNAse protection experiments reported here confirm the existence of two mRNAs for MAG which arise by alternative splicing of exon 12, as shown by Lai et al. Our results show that the p72MAG mRNA is expressed during the time of active myelin formation in the CNS, reaching a peak by post-natal day 22 and thereafter declining to adult levels by day 62. Conversely, p67MAG mRNA is produced as the minor species during myelin formation, but becomes the predominant form in adult brain. Cultures of oligodendrocytes express both forms of MAG. In the PNS, mRNA coding for p67MAG is predominant throughout development, reaching peak levels at day 6-10, whereas p72MAG mRNA is a very minor species. Alternative splicing also occurs at the 5' terminus. One form of mRNA lacking exon 2 from the 5' non-coding region is predominant in PNS, whereas mRNA containing exon 2 predominates in the CNS. Therefore, at least two and possibly four different mRNA species encode MAG. These results confirm the hypothesis that the two forms of MAG (p67 and p72) are generated by alternative splicing and show that each form is differentially regulated during development in the CNS and PNS.

Isolation and characterization of the human catalase gene.

Catalase is a tetrameric hemoprotein which degrades H2O2. Recombinant phage clones containing the human catalase gene have been isolated and characterized. The gene is 34 kb long and is split into 13 exons. The precise size and location of the exons has been determined. In addition, essentially full length catalase cDNA clones have been isolated and sequenced and used to tentatively identify the 5'-end of the gene. This assignment, if correct, predicts that the region upstream of the gene does not contain a TATA box. This region is GC rich (67%) and contains several CCAAT and GGGCGG sequences which may form part of the promoter. Translation of the catalase mRNA appears to begin immediately upstream of the amino-terminal Ala residue of catalase.