MBP and PLP are major structural protein components of myelin. Both proteins play a functional role in formation of myelin sheath and in maintenance of its compaction. Immune responses to MBP and PLP have been implicated in the pathogenesis of multiple sclerosis (MS), an auto-immune disease of the central nervous system. Recombinant forms of both proteins isolated and purified from bacterial or insect cell systems are commonly used to study the specificity of auto-response in MS. We have prepared recombinant forms of MBP and PLP stably expressed in CHO cells. Several clones with proper cytoplasmic MBP or surface PLP localization were obtained and characterized by flow cytometry and indirect immunostaining. CHO cells expressing the recombinant forms of MBP and PLP can be very useful in studies on the autoimmune mechanism of MS.

Summary Glycophorin C (GPC) and glycophorin D (GPD) are minor but important components of human RBC membranes. They carry the high‐frequency antigens Ge2, Ge3 and Ge4 of the Gerbich blood group system. The epitopes for five new monoclonal antibodies (MoAbs) with anti‐GPC specificity were characterized. Two antibodies (4G11 and 5B11) reacted with glycosylated N‐terminal epitopes, and three reacted with internal epitopes of GPC. Pepscan analysis showed that the MoAb RB11 required for binding the EPDP sequence, occurring twice in GPC polypeptide chain. The MoAb 7F11 recognized the sequence 13PLSLEPDP20, and the MoAb RB8 did not react with synthetic peptides. Further characterization of the internal epitopes was performed in fluorescence‐activated cell sorter (FACS) with the use of recombinant GPC and its variant forms transiently expressed on COS‐7 cells. The results indicated that the MoAb RB11 recognized distinctly its target sequence EPDP only in a normal GPC molecule. The reactivity of the MoAb 7F11 with the PLSLEPDP sequence was confirmed and found to be enhanced by the O‐glycan at the Ser15 residue. The MoAb RB8 recognized the glycopeptidic epitope in proximity to the Ser15 residue, requiring the presence of O‐glycan. The combination of immunochemical techniques with the use of the recombinant forms of GPC has made it possible to define the role of sugar chains in the recognition of peptidic epitopes in glycosylated antigen and sheds new light on the Gerbich system antigens.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

The epitopes for three murine anti-glycophorin C monoclonal antibodies 2G11, 3C1 and 3C4, previously characterized by Pepscan analysis, were analyzed by binding to recombinant normal and variant forms of GPC, expressed on COS7 cells.The epitope for the MoAb 2G11 consisted of aa residues (16)LEPDPGMA(23), with Leu16 as a key residue, and exposure of this epitope in GPC was dependent on glycosylation of Ser15. The MoAb 3C1 reacted with the short sequence of aa residues (EPDP39)-E-36 of GPC polypeptide chain and did not recognize the sequence (EPDP20)-E-17, partly due to glycosylation of Ser15. The MoAb 3C4 recognized the conformational epitope located within aa residues 36-49, and its binding was not dependent on glycosylation of Ser42.

The specificities of two murine anti‐Mg monoclonal IgG1 antibodies, 3B10 and 2D5, were determined by pepscan analysis. The peptides which correspond to various fragments of amino‐terminal portions of glycophorin A of group M (GPA‐M), N (GPA‐N) and Mg (GPA‐Mg), and replacement analogues of some of these peptides, were synthesized on plastic pins and tested for binding of the antibodies. Both antibodies bound strongly to the N‐terminal Mg octapeptide 1LSTNEVAM8, but they showed different subspecificities. The essential fragment of the epitope 2D5 are amino acid residues 2STNEV6. Replacement of any of these amino acid residues by Ala, and replacement of Glu5 residue by Gly, abolished or strongly reduced the antibody binding, but replacement of Asn4 by Thr gave only a moderate decrease of peptide activity. In contrast, the Leu1 and Asn4 residues were most essential components of the epitope 3B10, while Ser2, Thr3 and Glu5 seemed to be less important. Our present results and earlier ones on the specificity of human anti‐Mg alloantibodies and monoclonal anti‐M/Mg antibodies showed that antibodies reacting with Mg antigen recognize different fragments and/or different amino acid residues of the amino‐ terminal nonglycosylated domain of GPA‐Mg. The knowledge of fine specificities of antibodies reacting with Mg antigen is interesting in view of the presence of anti‐Mg alloantibodies in 1–2% of human sera.

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers, Evidence for homodimer formation has come primarily from radiation inactivation experiments, We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2, We stably transfected CHO cells with myc epitope-tagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal, We then sought evidence for dimer formation between the two forms of GalNAcT, Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT, As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence, The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER, Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.

A number of Golgi glycosyltransferases has been cloned to date. They all are membrane proteins and share the same type II topology, but they do not possess an obvious sequence homology which would suggest a common Golgi retention signal. However, it was shown that the membrane-spanning domain and its flanking regions contain necessary and sufficient information for Golgi retention of these enzymes. Currently, two mutually complementary models have been proposed to explain the mechanism of Golgi retention of glycosyltransferases mediated by their transmembrane domain. The first model postulates the retention through oligomerization, which prevents enzymes from entering the transport vesicles. The second suggests that retention depends on the length of a membrane-spanning domain and thickness of the membrane along the Golgi complex. It has to be pointed out that neither the oligomerization nor the membrane thickness model alone can answer all questions and further work is still needed to elucidate the retention process of Golgi proteins.

The peptidic epitopes of 12 anti-GPA and 4 anti-GPC antibodies were identified with the use of peptides synthesized on the pins. Most of the antibodies were specific for epitopes located in extracellular portion of glycophorins, and only 2 anti-GPA and 1 anti-GPC recognized epitopes in their C-terminal cytoplasmic tails. The extracellular GPA epitopes were located in two regions of the polypeptide chain, within a.a. residues 38-44 and 49-58.

Many Golgi membrane-bound glycosyltransferases are released from cells in a soluble form. To characterize this release process, we stably transfected Chinese hamster ovary cells with three myc epitope-tagged forms of cloned beta 1,4-N-acetylgalactosaminyltransferase (GalNAcT); two of these forms resided in the Golgi, while the third was retained in the ER, GalNAcT was released into the culture medium from cells transfected with the Golgi forms but not with the ER form of the enzyme. The medium from cells transfected with the Golgi forms contained disulfide-bonded dimers of GalNAcT, which carried neuraminidase sensitive, complex N-linked carbohydrate chains. This soluble species represented the major degradation product of cellular GalNAcT, which turned over with a half-time of about 1.7 h. The soluble species consisted of a mixture of truncated GalNAcT molecules, the major form of which was produced by cleavage near the boundary between the transmembrane and lumenal domains between Leu-23 and Tyr-24. This cleavage site fits the sequence pattern for sites cleaved by cathepsin D (van Noort, J.M., and van der Drift, A.C.M. (1989) J. Biol. Chem. 264, 14159-14164), These findings suggest that GalNAcT is converted from a membrane-bound to a soluble form as a result of cleavage by a cathepsin D-like protease in a compartment late in the Golgi secretory pathway.

Some monoclonal antibodies (MoAbs) directed against blood group M- related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti- M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser<-->Leu1 or Gly<-->Glu5 (M v N) and Thr<-->Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid- dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes.

Earlier studies reached conflicting conclusions as to the ability of the beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesizes gangliosides GM2 and GD2 to also produce gangliotriosylceramide (GA2). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain ganglioside GM3 as the most complex glycosphingolipid (GSL), whereas the CHO glycosylation mutant Lec2, which is deficient in sialylation, accumulates lactosylceramide with little GM3 being produced. We transfected both cell types with a plasmid containing a cloned GalNAc-T. Whereas transfected CHO cells produced GM2 as the major complex GSL, the major product in transfected Lec2 cells was GA2. Both types of transfected cells but not the untransfected cells expressed the transfected gene and contained high levels of enzyme activity for synthesizing both GM2 and GA2 in vitro. In summary, these results indicate that this enzyme can in fact synthesize GA2 as well as GM2 and GD2. In addition, these findings suggest that in CHO cells the synthesis of GM3 in vivo has priority over GA2 synthesis for utilization of the substrate lactosylceramide, resulting in little GA2 being produced even though GalNAc-T is present and active. Thus, competition for substrate between glycosylation pathways may have profound effects on the GSL pattern of cells.

The mouse monoclonal antibody M2A1 of IgG1 class, which is highly specific for blood group M antigen, was obtained and characterized by means of hemagglutination, enzyme‐linked immunosorbent assay, immunoblotting, and inhibition assays. The use of modified M glycoprotein preparations for inhibition tests and of variant M C N and Henshaw red cell membranes for immunoblotting showed that M2A1 recognized an epitope including the NH 2 ‐terminal serine and sialic acid residues of glycophorin A, whereas the fifth glycine residue was not involved. The reactivity of the antibody with M antigen was distinctly dependent on ionic strength and pH; the optimum was at pH 8 to 9. The α‐amino group of terminal serine residue was not necessary for the reaction with M2A1 antibody, and the results obtained suggested that the positive charge of this group contributed to decreasing antigen‐antibody reactions at pH below 8. The reaction of the antibody with blood group N antigen was not detectable in any of the assays used.

In a patient with myelodysplastic syndrom complement fixing IgG1 anti-Ge three weeks after Ge positive serologically compatible blood transfusion was found. The patient and one of his children were Ge-1, -2, -3. Red cells were elliptocytic. The patient's nonconsanguineous wife and other family members were Ge positive. In the red cell membranes of Ge negative persons glycophorin C was lacking. Abnormal glycophorin was present in the red cell membranes of all family members. Anti-Ge appeared to be of no clinical significance. Transfusion of serologically incompatible Ge positive blood provided beneficial effect. After this transfusion anti-Ge disappeared from the serum and was not detected at the whole follow-up, although the patient received Ge positive blood several times. Immunological tolerance towards Ge antigen is suggested.

Glycophorins of human erythrocytes have been extensively studied and the structure of three of them is fully (glycophorins A and C) or almost fully (glycophorin B) known [1, 2]. Glycophorins span the erythrocyte membrane and their NH 2 -terminal domains exposed at the cell surface are heavily glycosylated. Glycophorin A occurs in two genetically determined forms carrying at NH 2 -terminal end blood group M and N antigenic determinants. Glycophorin B (blood group Ss glycoprotein) has the structure of NH 2 -terminal region (a.a. residues 1–26) identical to glycophorin A of blood type N, and also shows a high degree of homology with glycophorin A in the internal portion of the molecule, whereas glycophorin C has a different amino acid sequence. The knowledge of structure and orientation in the membrane and genetic differentiation of glycophorins facilitate elucidation of the fine specificity of anti-glycophorin antibodies. The 30 anti-glycophorin-antibodies obtained were tested by agglutination of untreated and modified erythrocytes, immunoblotting, and binding to glycophorin A in microtiter plate ELISA. Moreover, inhibition of antibodies by untreated and modified glycophorin A preparations was studied. The methods used were described in detail in our recent publications [3, 5]. The antibodies could be divided into groups (Table I) , depending on specificity. The 19 antibodies recognized epitopes located at the NH 2 -terminal end of glycophorin A that could be easily shown by specific or distinctly preferable reactivity with blood group M (8 MoAbs) or N (11 MoAbs) antigen. The antibodies with anti-N specificity also reacted with glycophorin B. Among the remaining blood group MN-unrelated antibodies, 4 were specific for glycophorin A, 3 recognized epitopes common for glycophorins A and B, 2 reacted to glycophorin C, and the specificity of 2 antibodies could not be clearly established. The antibodies in each group differed in sub-specificity and antigen-binding properties.