Samar K. Kundu

Antibodies to glycosphingolipids in patients with multiple sclerosis and SLE.

We used a liposome lysis assay to measure antibodies against a panel of glycolipids. Antibodies to one or more compounds were detected in 34 of 46 patients with multiple sclerosis, 19 of 31 patients with systemic lupus erythematosus (SLE), and in the majority of patients with cranial trauma or cerebrovascular accidents. Antibodies against ganglioside GM1 and asialo GM1 were found most commonly, and they were frequently present in the same sera. The specificity of the antibodies was tested in four sera that contained antibodies to both glycolipids. The anti-GM1 antibodies cross-reacted with asialo GM1, but the converse was not true. Among patients whose sera contained antibodies to glycolipids, anti-asialo GM1 alone was more common in patients with SLE (7 of 17) than in multiple sclerosis (2 of 34; p = 0.004). Anti-GM1 alone was found in 9 of 34 patients with multiple sclerosis and 1 of 17 patients with SLE, a difference that was not statistically significant (0.135). No correlation was observed between the presence of anti-glycolipid antibodies and symptoms related to the nervous system in patients with SLE. Because of our inability to detect these antibodies by a solid phase immunoassay (ELISA), a comparison was made of the titers obtained with three monoclonal anti-glycolipid antibodies in the liposome lysis assay and ELISA. The ELISA was less sensitive in all instances, requiring from four to 1000 times as much antibody as the liposome lysis assay to give a positive test. We conclude that antibodies to glycolipids occur frequently in patients with multiple sclerosis, SLE, major cranial trauma, and cerebrovascular accidents. Their role in the initiation or perpetuation of inflammatory disease of the central nervous system has yet to be determined.

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.

Binding of momoclonal antibody A2B5 to gangliosides

Monoclonal antibody A2B5 (Eisenbarth et al, Proc. Nat. Acad. Sci. (1979, 76:4913-4917), which reacts with neurons, thymic epithelium and peptide-hormone secreting cells of several species, was reported to react specifically with brain tetrasialogangliosides. We have found that A2B5 binds to gangliosides GQ1b, GD3, GD2, disialolactoneotetraosylceramide, and probably to GT1a, when assayed by an immunostaining procedure that detects binding of antibody to gangliosides on a thin-layer plate. Additional data obtained by complement fixation revealed that this antibody reacted most strongly with ganglioside GQ1b almost as well with disialogangliosides GD3, GD2 and disialolactoneotetraosylceramide, weakly with GD1b and GT1b, and very weakly with GM3 and GD1a. These data indicate that A2B5 cannot be regarded as a specific reagent for the recognition of tetrasialogangliosides.

Rapid separation of gangliosides by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) procedure for the rapid separation of individual ganglioside components on a 5-micron porous silica gel column using a mixture of isopropanol-hexane-water with increasing water content and decreasing hexane content. Total ganglioside mixtures were first fractionated on DEAE-silica gel according to the number of sialic acid residues. Each fraction was then separated into individual ganglioside components by HPLC. Total elution time was less than 2 h. This procedure has been applied for the separation of major ganglioside components of human erythrocytes and beef brain and is highly reproducible.

Spontaneous and plasma factor-mediated transfer of pyrenyl cerebrosides between model and native lipoproteins

A series of pyrenyl glucocerebrosides was synthesized by reacylation of psychosine with pyrene-labeled fatty acids having 3-11 methylene units. When incorporated into model high-density lipoproteins consisting of dimyristoylphosphatidylcholine-apolipoprotein A-II complexes and incubated with unlabeled complexes, these lipids exhibited spontaneous transfer. Half times of transfer varied from 1.5 min to 365 min at 37 degrees C. The logarithm of the rate of transfer was linearly related to the number of fatty acyl methylene units and HPLC retention time. Transfer occurred by passage of lipid monomers through the aqueous phase. Spontaneous transfer of the glycolipids also occurred when they were incorporated into native high-density lipoproteins. Rates of transfer between native high-density lipoprotein particles were higher than those observed between model high-density lipoprotein particles. A partially purified lipid exchange protein from plasma, as well as unfractionated lipoprotein-deficient serum, stimulated the transfer of fluorescent glycolipid between model high-density lipoprotein or native high-density lipoprotein and low-density lipoprotein 2-24 fold. The protein also stimulated the transfer of tritiated ganglioside GM3 between native low-density lipoprotein and high-density lipoprotein. This protein may play a role in glycolipid exchange in vivo.

Glycosphingolipid receptors for anti-Gd and anti-p cold agglutinins

Anti-Gd and anti-p cold agglutinins exhibit similar serological properties: neuraminidase treatment of erythrocytes greatly reduces their agglutinability by these antibodies and protease treatment enhances their agglutination. We reported previously that an anti-p cold agglutinin was inhibited by sialosyllactoneotetraosylceramide, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer, the most abundant ganglioside of human erythrocytes. We now report that two less abundant gangliosides are more potent inhibitors of this antibody, and of the anti-Gd antibodies, than sialosyllactoneotetraosylceramide. These two gangliosides have the same carbohydrate chain, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(SNH), but they differ in their ceramide moiety. The principal fatty acid of SNH-1 is C16:0, whereas SNH-2 contains a predominance of C22:0, C24:0 and C24:1. No inhibition was produced by the ganglioside, NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer. Another monoclonal cold agglutinin, Sa, which shares some serological properties with anti-Gd cold agglutinins, was not inhibited by any of these gangliosides.

Immunochemical Relationship Between Forssman and Globoside Glycolipid Antigens

The rabbit antibody response to the human blood group P glycolipid antigen, globoside, GalNAc(beta 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc-Cer, has been examined with respect to cross-reactions with the structurally related Forssman glycolipid GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc-Cer. Immunoadsorbent columns were used to isolate three purified antibody populations from the anti-globoside sera: (1) fraction A, antibodies that cross-react with both glycolipids; (2) fraction B, antibodies that react with Forssman antigen but not with globoside; and (3) fraction C, antibodies that are specific for globoside. The proportion of each fraction in the total antibody response to globoside appears to be related to preexisting immunity to these antigens. A rabbit with a high preimmune titer to Forssman antigen produced a large amount of Forssman-specific antibody, whereas a rabbit with a low or nonexistent preimmune titer of anti-Forssman antibody produced large amounts of globoside-specific antibody. The presence of Forssman-specific antibody in an immune response to globoside is an example of a heteroclitic type of immune response.

Stalosylglobotetraosylceramide: A marker for amyotropic lateral sclerosis☆

Gangliosides of healthy and pathologic muscles (amyotropic lateral sclerosis and facio-scapulo-humeral muscular dystrophy) were studied. Total ganglioside content of the affected muscles was approximately 2 fold higher than the unaffected muscles. Our results showed that ALS muscle contained a ganglioside which was absent in the unaffected and FSH muscular dystrophic muscles. Based on the results of hydrolysis with Vibrio cholerae neuraminidase and subsequent reactivity of the asialo derivative towards anti-globotetraosylceramide, we propose that the ALS ganglioside is sialosylglobtetraosylceramide, NeuAc(alpha 2-3)Ga1NAc(beta 1-3)Ga1(alpha 1-4)Ga1(beta 1-4)G1c-Cer.

Antibodies to Glycosphingolipids in Patients with Multiple Sclerosis

Antibodies to one or more glycosphingolipids were detected by means of a liposome lysis assay in the sera of 60/81 patients with multiple sclerosis, 24/42 patients with systemic lupus erythematosus and in the majority of patients with cranial trauma or cerebrovascular accidents. Antibodies against ganglioside GM1 and asialo GM1 were found most commonly and they were frequently present in the same sera. Among patients whose sera contained antibodies to glycolipids, anti-GM1 alone occurred more frequently in patients with multiple sclerosis (14/59) than in systemic lupus erythematosus (1/22; p = 0.045) and antiasialo GM1 alone was more common in patients with lupus (9/22) than in patients with multiple sclerosis (8/59, p = 0.007). In 10 sera analyzed, all of the antibodies against these two glycolipids were of the IgM class, and some fluctuation in antibody titers was noted over a three-month period. The role of these antibodies in the initiation or perpetuation of inflammatory diseases of the central nervous system remains to be determined.

Muscle glycolipids in inherited muscular dystrophy of chickens

Gangliosides and neutral glycolipids of muscles from normal and dystrophic chickens were studied. Total glycolipid content of the degenerating muscles was higher than the normal muscles. In addition, the myopathic muscles contained a ganglioside which was absent in the unaffected muscles from normal and dystrophic chickens. Based on the thin‐layer chromatographic mobility, treatment with neuraminidases from Vibrio cholerae and Arthrobacter ureafaciens, and reactivity of the asialo‐derivative towards anti‐ganglio‐N‐triaosylceramide antibody, the dystrophic‐specific ganglioside was tentatively identified as GM2. Data obtained from young and old dystrophic chickens suggested a direct relationship of this ganglioside to muscular dystrophy.