Topology of Kell blood group protein and the expression of multiple antigens by transfected cells

Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS- 1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.

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Topology of Kell blood group protein and the expression of multiple antigens by transfected cells

Blood ◽

10.1182/blood.v84.10.3518.3518 ◽

1994 ◽

Vol 84 (10) ◽

pp. 3518-3523 ◽

Cited By ~ 21

Author(s):

DC Russo ◽

S Lee ◽

M Reid ◽

CM Redman

Keyword(s):

Amino Acid ◽

Blood Group ◽

Disulfide Bonds ◽

K562 Cells ◽

Amino Acid Residues ◽

Transfected Cells ◽

Human Red Blood Cells ◽

Antigenic Properties ◽

Group Protein ◽

Low Incidence

Abstract Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS- 1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.

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Anti-M monoclonal antibodies cross-reacting with variant Mg antigen: an example of modulation of antigenic properties of peptide by its glycosylation

Blood ◽

10.1182/blood.v84.7.2340.2340 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2340-2345 ◽

Cited By ~ 16

Author(s):

E Jaskiewicz ◽

M Czerwinski ◽

D Syper ◽

E Lisowska

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Blood Group ◽

Polypeptide Chain ◽

Protein Glycosylation ◽

Glycophorin A ◽

Amino Acid Residues ◽

Terminal Portion ◽

Peptide Analogs ◽

Antigenic Properties

Abstract 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.

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Anti-M monoclonal antibodies cross-reacting with variant Mg antigen: an example of modulation of antigenic properties of peptide by its glycosylation

Blood ◽

10.1182/blood.v84.7.2340.bloodjournal8472340 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2340-2345

Author(s):

E Jaskiewicz ◽

M Czerwinski ◽

D Syper ◽

E Lisowska

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Blood Group ◽

Polypeptide Chain ◽

Protein Glycosylation ◽

Glycophorin A ◽

Amino Acid Residues ◽

Terminal Portion ◽

Peptide Analogs ◽

Antigenic Properties

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.

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Cleavage of the peptide chain of the blood group substances at the hydroxy amino acid residues

Bulletin of the Academy of Sciences of the USSR Division of Chemical Science ◽

10.1007/bf00846021 ◽

1965 ◽

Vol 14 (12) ◽

pp. 2195-2195

Author(s):

V. A. Derevitskaya ◽

S. G. Kara-Murza ◽

N. K. Kochetkov

Keyword(s):

Amino Acid ◽

Blood Group ◽

Peptide Chain ◽

Amino Acid Residues ◽

Hydroxy Amino Acid ◽

Hydroxy Amino ◽

Blood Group Substances

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Influence of Blood Group and Secretor Status on Carbohydrate Structures in Human Gastric Mucins: Implications for Peptic Ulcer

Clinical Science ◽

10.1042/cs0890405 ◽

1995 ◽

Vol 89 (4) ◽

pp. 405-415 ◽

Cited By ~ 4

Author(s):

R. L. Sidebotham ◽

J. H. Baron ◽

J. Schrager ◽

J. Spencer ◽

J. R. Clamp ◽

...

Keyword(s):

Peptic Ulcer ◽

Amino Acid ◽

Blood Group ◽

Average Length ◽

Amino Acid Residues ◽

Secretor Status ◽

Increased Risk ◽

The Mean ◽

The Difference ◽

Carbohydrate Chains

1. The content and distribution of carbohydrate was examined in mucus glycopolypeptides from human antral mucosae. 2. The mean amount of carbohydrate per 1000 amino acid residues was found to be similar in glycopolypeptides with A, B or H activity. It was slightly, though significantly, less in glycopolypeptides lacking these determinants, because carbohydrate chains were of a shorter average length than in the A-, B- or H-active preparations. This difference was reflected in the sizes of oligosaccharide—alcohols released from representative glycopolypeptides with alkaline borohydride. 3. Differences between A-, B- or H-active and non-secretor glycopolypeptides in terms of the mean number of carbohydrate chains per 1000 amino acid residues were found to be small, and without significance. 4. The average number of peripheral monosaccharide units per 1000 amino acid residues was greater in A-active than in H-active, and least in non-secretor, glycopolypeptides. This order was reversed for monosaccharide units incorporated into skeletal (core plus backbone) structures. The difference in each case was statistically significant. 5. These findings suggest that the increased risk of peptic ulcer associated with blood group O and non-secretor status is unlikely to be attributable to an inherent deficiency in the protective mucus layer, linked to differences between mucins that are associated with A, B or H activity. Other hypotheses linked to infection with Helicobacter pylori are examined.

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Human red blood cell Wright antigens: a genetic and evolutionary perspective on glycophorin A-band 3 interaction

Blood ◽

10.1182/blood.v87.9.3942.bloodjournal8793942 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3942-3947 ◽

Cited By ~ 34

Author(s):

CH Huang ◽

ME Reid ◽

SS Xie ◽

OO Blumenfeld

Keyword(s):

Amino Acid ◽

Nonhuman Primates ◽

Antigen Expression ◽

Band 3 ◽

Structural Basis ◽

Antigenic Structure ◽

Glycophorin A ◽

Amino Acid Residues ◽

Protein Protein Interaction ◽

Human Red Blood Cells

The Wright (Wra/Wrb) blood group polymorphism is defined by an allelic change (Lys658Glu) in the band 3 protein; nevertheless, the Wrb antigen apparently requires glycophorin A (GPA) for surface presentation. To gain insight into the structural basis for this protein-protein interaction and delineate its relationship with Wrb antigen expression, we investigated GPA and band 3 sequence polymorphisms occurring in rare humans and nonhuman primates. The lack of GPA or amino acid residues 59 through 71 of GPA results in the absence of Wrb from human red blood cells (RBCs) exhibiting the MkMk, En(a-), or MiV phenotype. However, the SAT homozygous cells carried a Glu658 form of band 3 and a hybrid glycophorin with the entire GPA extramembrane domain from residues 1 through 71, yet expressed no Wrb antigen. This finding suggests that formation of the Wrb antigenic structure is dependent on protein folding and that the transmembrane junction of GPA is important in maintaining the required conformation. Comparative analyses of GPA and band 3 homologues led to the identification in the interacting regions of conserved and dispensable amino acid residues that correlated with the Wrb positive or negative status on nonhuman primates. In particular, the chimpanzee RBCs cells expressed Wrb and the Glu658 form of band 3, which is identical to humans, but their GPA contained the Gly rather than Arg residue at position 61. Taken together, the results suggest that (1) Arg61 of GPA and the proposed Arg61-Glu658 charge pair are not crucial for Wrb antigen exhibition and (2) the role of GPA for interaction with band 3, including Glu658, probably involves a number of amino acid residues located in the alpha-helical region and transmembrane junction.

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A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽

10.1182/blood.v93.3.918 ◽

1999 ◽

Vol 93 (3) ◽

pp. 918-924 ◽

Cited By ~ 19

Author(s):

Eileen Collins Tozer ◽

Elizabeth K. Baker ◽

Mark H. Ginsberg ◽

Joseph C. Loftus

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Single Amino Acid ◽

Chimeric Receptor ◽

Amino Acid Residues ◽

Genetic Approach ◽

Specific Amino Acid ◽

Transfected Cells ◽

Binding Function ◽

A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

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Identification of tau protein regions required for process formation in PC12 cells

Journal of Cell Science ◽

10.1242/jcs.107.12.3403 ◽

1994 ◽

Vol 107 (12) ◽

pp. 3403-3412 ◽

Cited By ~ 2

Author(s):

J.G. Leger ◽

R. Brandt ◽

G. Lee

Keyword(s):

Amino Acid ◽

Pc12 Cells ◽

Quantitative Difference ◽

Microtubule Assembly ◽

Amino Acid Residues ◽

Transfected Cells ◽

Process Formation ◽

Process Outgrowth ◽

In Cells

Tau is a neuronal microtubule-associated protein that is required for the development and maintenance of neuronal cell polarity. It promotes microtubule assembly in vitro and we have recently reported that a specific tau region, which spans amino acid residues 154–172 of human fetal tau, is not required for growth of existing microtubules, but is required for nucleation of new microtubules. These residues also confer stronger microtubule binding activity in 3T3 cells. The aim of this study was to investigate the functional organization of tau in relation to its role in promoting process formation in a neuronal model system. We transfected undifferentiated PC12 cells with vectors expressing tau fragments and treated the expressing cells with cytochalasin B to allow process extension. We found that deletion of amino acid residues 154–172 greatly reduced the percentage of transfected cells bearing processes compared to that of cells transfected with full-length tau or with an amino-terminally deleted tau fragment containing residues 154–172. These differences do not appear to result from a quantitative difference in protein expression, as shown by immunoblot analysis of transfected cells. We also observed that while the presence of tau fragments increases acetylation of microtubules, the pattern of acetylation in cells transfected with the fragment missing residues 154–172 is less extensive, suggesting that it does not result in the same level of stabilization as the longer tau fragments. Taxol promoted process outgrowth in cells treated with cytochalasin and restored process outgrowth to cells transfected with the tau fragment lacking this activity. Therefore, process formation involves primarily the stabilization and nucleation of microtubules. We conclude that the residues necessary for conferring microtubule nucleation activity of tau in vitro are important for process formation in vivo. It is likely that these residues influence the binding affinity and therefore the stabilization activity of tau.

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A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽

10.1182/blood.v93.3.918.403k26_918_924 ◽

1999 ◽

Vol 93 (3) ◽

pp. 918-924 ◽

Cited By ~ 2

Author(s):

Eileen Collins Tozer ◽

Elizabeth K. Baker ◽

Mark H. Ginsberg ◽

Joseph C. Loftus

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Single Amino Acid ◽

Chimeric Receptor ◽

Amino Acid Residues ◽

Genetic Approach ◽

Specific Amino Acid ◽

Transfected Cells ◽

Binding Function ◽

A Cell

An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

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