The effect of cysteine modification and proteinases on the major antigens (D, C, c, E and e) of the Rh blood group system.

We have confirmed and extended previous observations showing that the (Rh) D antigen of erythrocyte membranes is destroyed by various reagents that modify cysteine (Cys) residues (Res.) and by trypsin as well as chymotrypsin, using thirty examples of monoclonal or polyclonal anti-D in heamglutination inhibition assays. We have also shown that most C, c, E, e and BS58 epitopes are inactivated or weakened by most Cys reagents and by these proteinases, using monoclonal and polyclonal antibodies. Inactivation by 5,5-dithiobis-(2-nitrobenzoic acid) was always fully reversible after subsequent dithioerythritol treatment. The essential Cys Res. appear to be buried in the membrane in view of the inability of some reagents to inactivate (iodoacetamide, iodoacetic acid) or reactivate (reduced glutathione) the antigens. Data obtained with N-ethylmaleimide indicate that inactivation of the C and c antigens is, at least in part, attributable to (a) Cys Res. that is (are) different from that (those) involved in the E and e antigens. Data obtained with the Cys reagents and the proteinases suggest that more than one peptide loop of the Rh proteins is involved in the major Rh antigens.

Characterization of new murine monoclonal antibodies directed against glycophorins C and D.

Six new murine monoclonal antibodies (mAbs) directed to the erythrocyte membrane glycophorins C (GPC) and D (GPD) were obtained from splenocytes of different BALB/c mice immunized with human red blood cells, and fully characterized. The mAbs were selected by agglutination tests with control and Gerbich-negative cells, and by immunoblotting analysis. They showed specificity for the N-terminal domain(s) of GPC (and GPD) and were classified into three categories by competitive analysis using 125I-labelled antibodies and real-time biospecific interaction. The first group (NaM10-7G11, NaM70-1G4 and NaM77-7B6) compete for epitope(s) located at the N-terminal portion of GPC. Agglutination-inhibition tests revealed that the 7G11 epitope involves the amino group of Met1 and sialic acid residue(s) whereas the 1G4 and 7B6 epitopes contain O-glycans. NaM89-2G11 belongs to a second group; its epitope is located in a region including Glu17, Asp19 and (an) O-glycan(s). The third group comprises mAbs NaM19-3C4 and NaM98-3C1 which bind to both GPC and GPD in proximity of the binding site of human anti-Ge:3 antibodies. In addition, mAb 3C4 (anti-GPC/GPD) was found to bind to approximately 125,000 sites per red cell. Considering that the ratio of the GPC to GPD is about 3-4 to 1, the number of GPC and GPD molecules was estimated as 95,000 and 35,000, respectively.

[Monoclonal antibodies in blood group serology. Part 1: Technique of production, ABO and Lewis system]. / Monoklonale Antikörper in der Blutgruppenserologie. Teil 1: Technik der Herstellung, AB0- und Lewis-System.

OBJECTIVE: The introduction of monoclonal antibodies in routine blood group typing has also given rise to new findings. This paper was designed to demonstrate and clarify modifications relevant in practice. SOURCES: Our own experience in the production and application of monoclonal antibodies as well as the relevant literature are taken into consideration. RESULTS: The development and production of murine and human monoclonal antibodies is described especially regarding the selection and the safety of clones. An update of the availability and use of monoclonal antibodies in respect to the different blood group systems is given, with special reference to new findings like the A1(B) and B(A) phenomena. CONCLUSION: The application of monoclonal antibodies in blood group serology has improved the detection of weak variants and the availability of these reagents in general. Further improvements in this technology will make monoclonal antibodies also available for other blood group systems.

[Monoclonal antibodies in blood group serology. Part 2: MNS, Rh and other blood group systems]. / Monoklonale Antikörper in der Blutgruppenserologie. Teil 2: MNS-, Rh- und übrige Systeme.

OBJECTIVE: The introduction of monoclonal antibodies in routine blood group typing has also given rise to new findings. This paper was designed to demonstrate and clarify modifications relevant in practice. SOURCES: Our own experience in the production and application of monoclonal antibodies as well as the relevant literature are taken into consideration. RESULTS: The development and production of murine and human monoclonal antibodies is described especially regarding the selection and the safety of clones. An update of availability and use of monoclonal antibodies in respect to the different blood group systems is given, with special reference to the reactivity of these new reagents with weak D-positive (Du) red blood cells. CONCLUSION: The application of monoclonal antibodies in blood group serology has improved the detection of weak variants and the availability of these reagents in general. Further improvements in this technology will make monoclonal antibodies also available for other blood group systems.

Miltenberger subsystem of the MNSs blood group system. Review and outlook.

The Miltenberger (Mi) classes represent a group of phenotypes for red cells that carry low frequency antigens associated with the MNSs blood group system. The antigens of this system are known to be located on two sialoglycoproteins denoted as glycophorin A (GP A) and GP B. The structural alterations of seven (classes I, II, III, V, VI, VII, VIII) Mi variants and a related variant (J.L.) have been elucidated. Based on these data and yet incomplete studies of the Mi antigens, the approximate structural alterations in class IV and IX may be predicted. In addition, knowledge of the various structures and partial characterization of the Mi antigens allows one to propose detailed hypotheses concerning the epitopes recognized by the various antibodies that define the Mi subsystem. The understanding of the Mi subsystem at the molecular level paves the way for future studies aimed at a more detailed elucidation of epitopes of Mi-related antibodies, the characterization of novel Mi variants and a search for hypothetical, hitherto unknown Mi-related antibodies.

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

The Mz variety of the St(a+) phenotype--a variant of glycophorin A exhibiting a deletion.

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)

High-frequency antigens of human erythrocyte membrane sialoglycoproteins. VI. Monoclonal antibodies reacting with the N-terminal domain of glycophorin C.

The epitopes of seven mouse monoclonal antibodies which are related to the Gerbich blood group system were investigated. BRIC4, BRIC10, GERO and MR4-130 have been published earlier. The three others (APO1, APO2, APO3) were prepared by immunization with normal human erythrocytes and detected by screening with red blood cells that lack glycophorins C and D. Using immunoblotting and hemagglutination inhibition assays, the epitopes for all antibodies were found to be located on glycophorin C. Hemagglutination inhibition experiments with peptides and chemically modified glycophorins revealed that MR4-130, GERO, BRIC10 and APO2 are all directed against identical or rather similar epitopes comprising the N-terminal three or four residues of glycophorin C. Modification of the N-terminal methionine residue or release of sialic acid attached to oligosaccharide(s) at the third and/or fourth position(s) destroyed all these antigens. The epitope of APO3 was found to comprise glutamic acid17 and/or aspartic acid19 as well as the oligosaccharide attached to serine15. The antigens of BRIC4 and APO1 were found to be located within the residues 2-21 and to comprise sialic acid attached to O-glycosidically linked oligosaccharide(s). However, these epitopes could not be elucidated further. Radio-iodinated MR4-130 bound to 39,000 receptor sites per normal red blood cell. Binding of the labelled antibody was completely inhibited by unlabelled MR4-130, BRIC10, APO2 and GERO. APO1 caused partial inhibition suggesting that it is directed against an adjacent site. BRIC4, APO3 and anti-Ge3 did not inhibit the binding of labelled MR4-130 to any significant extent.

Structural analysis of glycophorin A from Miltenberger class VIII erythrocytes.

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the erythrocytes of two individuals heterozygous for the Mi-VIII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic glycopetide from glycophorin A comprising the residues 40-61 was deduced from automated and manual sequence analyses. The Mi-VIII-specific glycophorin A was found to exhibit an arginine----threonine exchange at position 49. The threonine residue was found to be glycosylated. Hemagglutination and hemagglutination inhibition assays demonstrated that one of the Mi-VIII-characteristic antigenic determinants (Anek) is located within the residues 40-61 of glycophorin A. Furthermore, erythrocytes from the two Mi-VIII heterozygotes reacted only weakly with anti-EnaKTsera, suggesting that the Mi-VIII-specific glycophorin A does not express the EnaKT antigen that is located within the positions 46-56 of normal glycophorin A. Our data suggest that the Mi-VIII-specific glycophorin A represents the evolutionary link between normal glycophorin A and the Mi-VIII-specific molecule which exhibits arginine----threonine and tyrosine----serine exchanges at the positions 49 and 52, respectively. Our data also provide an explanation for the close serological similarity between Mi-VII and Mi-VIII erythrocytes.

Structural homology between glycophorins C and D of human erythrocytes.

Glycophorin C (GPC) and D (GPD) are minor glycoproteins which are believed to be important for the structural integrity of the red cell membrane. We have investigated the structural relationship between these glycoproteins by both immunological and structural investigations: 1. A rabbit anti-serum produced against GPD reacts strongly with GPC and the abnormal glycoproteins of Gerbich: -2, -3 and Gerbich: -2,3 red cells, and recognizes most probably the homologous C-terminal portions of GPC and GPD. The two molecules however differ at their N-terminus. 2. One-dimensional mapping of the peptides obtained after tryptic, chymotryptic, V8 protease or acid cleavage of 125I-labelled GPC and GPD, indicated that GPC and GPD are structurally related but some differences were found indicating that additional peptides were generated from GPC. 3. The partial primary structure of GPD was determined. The sequencing data are consistent with the assumption that GPD represents an abridged version of GPC that comprises residues approximately 21/29-128 and exhibits a N-terminal residue that is blocked by an as yet undefined group.

A novel variety of the Dantu gene complex (DantuMD) detected in a Caucasian.

The first Caucasian (MD) shown to exhibit the low-frequency MNSs system antigen, Dantu was detected due to an increased tendency of erythrocytes to be aggregated by substances that promote red cell agglutination. The donor was found to exhibit a novel variety of the Dantu gene complex (DantuMD), as judged from biochemical, immunochemical, and serological studies. The glycophorin (GP) A level of MD's erythrocyte membranes were slightly decreased (about 17%) but GP B was not significantly different from normal. GP A and GP B of MD's cells were shown to carry M and N or S and s antigens, respectively, indicating that MD exhibits two genes encoding GP A and two genes encoding GP B. MD's cells contain a Dantu-, N- and s-specific GP B-GP A hybrid GP (molar ratio to GP A approx. 0.6:1.0). Partial amino-acid sequence analysis indicates that the structure of this molecule is rather similar to, or completely identical with, that of the hybrid GP in DantuNE erythrocytes. The residues 1-39 or 40-99 of the latter molecule correspond to the residues 1-39 of s-specific GP B and the residues 72-131 of GP A, respectively. Statistical evidence suggests that MD exhibits a single gene encoding the hybrid GP. Thus, MD appears to be heterozygous for a typical anti-Lepore type gene complex that seems to comprise genes for GP A, GP B, and the GP B-GP A hybrid. The diminished GP A level and a decreased galactose-oxidase labelling of the major membrane protein (anion channel protein, band 3) in MD's cells is in accordance with previous data suggesting that band 3 might form a complex with GP A and the Dantu-specific hybrid GP. This complex formation may be necessary for optimum incorporation of the latter molecules into the membrane.

MNSs and Gerbich blood group systems.

Human red blood cell membranes contain four sialic acid-rich glycoproteins, denoted as glycophorins (GPs), that carry the antigens of the MNSs and Gerbich (Ge) blood group systems. The MNSs locus corresponds to two related and adjacent genes that encode the polypeptide sequences of two of these molecules: GP A and GP B. The structural differences between the major polymorphic antigens M and N or S and s are determined by amino acid heterogeneities within the glycosylated NH2-terminal domain of GP A or GP B, respectively. Because the NH2-terminal 26 residues of GP B are identical with those of GP A possessing blood group N specificity, the former molecule carries an additional N antigen, denoted as 'N'. Apart from the major antigens, GP A and GP B carry several high- or low-frequency receptors that are encoded by the MNSs locus. Additional alleles are apparently silent or produce GP A-GP B hybrid molecules. The Ge locus is similar to the MNSs locus in that it appears to correspond to the adjacent genes encoding the polypeptide chains of GP C and GP D. However, the Ge system does not include antigens that are polymorphic in Caucasoids. Because all GPs are heavily glycosylated, oligosaccharides, in addition to protein, are involved in antigens of the MNSs and Ge systems. The carbohydrate units on all GPs account for additional antigens that are not part of the MNSs or Ge systems.

Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D), (c), and (E) antigens are carried by distinct polypeptide chains.

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P-L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one-dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.

High frequency antigens of human erythrocyte membrane sialoglycoproteins, V. Characterization of the Gerbich blood group antigens: Ge2 and Ge3.

The molecular properties of the major, high-frequency antigens (Ge2 and Ge3) of the human Gerbich blood group system were investigated using 14 different alloantibodies from rare Ge: -1,-2,-3 or Ge: -1,-2,3 individuals. Various modification, fractionation or fragmentation products of glycophorins (sialoglycoproteins) from normal erythrocytes (phenotype Ge: 1,2,3) were used in hemagglutination inhibition assays. The location of the antigens was also studied by blotting of proteins, separated by dodecyl sulfate polyacrylamide gel electrophoresis, to nitrocellulose and detection of bound antibodies by 125I-labelled protein G. Anti-Ge3 was found to be directed against a region of glycophorin C that surrounds a tryptic cleavage site at position 48 and a similar region of glycophorin D whose structure is not yet known. NeuAc residue(s), probably representing part(s) of a carbohydrate unit attached to serine42 of glycophorin C, methionine, aspartic or glutamic acid, tryptophan and/or arginine residue(s) are involved in the Ge3 epitopes, as judged from chemical modification. The Ge2 epitopes were found to be located on a tryptic glycopeptide from glycophorin D comprising about 20-30 amino-acid residues. NeuAc residue(s), attached to serine-/threonine-linked oligosaccharide(s), are involved in the Ge2 determinants. Using the immunoblotting technique, it could also be shown that the 'new' glycophorin in Ge: -1,-2,3 cells carries the Ge3 antigen.

Hybrid glycophorins from human erythrocyte membranes. Isolation and complete structural analysis of the novel sialoglycoprotein from St(a+) red cells.

Human red cells from donor Pj carry the Sta blood group antigen and an unusual sialoglycoprotein of 24 kDa molecular mass tentatively identified as a hybrid molecule of the anti-Lepore type [Blanchard et al. (1982) Biochem. J. 203, 419-426]. This component is resistant towards proteinase treatment and was purified from trypsin-treated and chymotrypsin-treated Pj erythrocytes. The molecule is composed of 99 amino acid residues whose alignment was established following manual and automatic sequencing of cyanogen bromide, trypsin, chymotrypsin and V8 proteinase peptides. The polypeptide chain comprises residues 1-26/28 of glycophorin B and residues 59/61-131 of glycophorin A. The sugar composition resembles that of glycophorin B, indicating the absence of an N-glycosidic chain. Identical sequences were obtained from analyses of the 24-kDa component purified from unrelated St(a+) donors. These results support the hypothesis that glycoprotein Pj represents a B-A hybrid molecule which is encoded by a new gene product resulting from an unequal crossing-over between the genes coding for the polypeptide chains of the glycophorins A and B. The novel molecule carries both N and Sta blood group antigens. The N activity is clearly understandable from the sequence of the five N-terminal residues (Leu and Glu at positions 1 and 5 respectively). Inhibition studies with the untreated and chemically modified hybrid glycoprotein indicate that the Sta determinant is located within residues approximately 25-30 of the molecule, which corresponds to the newly formed sequence found neither in glycophorin A nor in glycophorin B.