Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.

The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293 K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4-8 mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25 A resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75 A resolution. Data were collected that indicated that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 77.1, c = 118.1 A and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP).

Antigenic properties of human glycophorins--an update.

Glycophorins are complex heavily glycosylated antigens carrying peptidic and glycopeptidic epitopes. Detailed immunochemical studies showed that GPA/GPB and GPC/GPD molecules have defined sites which are particularly immunogenic. These sites include N-terminal portions of all glycophorins, internal fragments of their extracellular domains, and cytoplasmic tails. The extracellular epitopes involve directly oligosaccharide chains (e.g. blood group M- and N-related epitopes, or N-terminal epitopes of GPC) or have peptidic character, shown by the reaction of respective antibodies with synthetic peptides. Peptidic eitopes are independent of glycosylation, or are variably affected by adjacent O-glycans which may mask the epitopes or may be required for a proper exposure of an antibody binding site. Several low incidence epitopes are present on variant glycophorin molecules. Among anti-glycophorin antibodies there are the 'bispecific' ones, or antibodies recognizing an epitope formed by an interaction of two proteins (Wr(b)). Alltogether, the glycophorins serve as convenient model antigens for studying Ag-Ab interaction and a role of O-glycosylation in protein antigenic properties. Moreover, well defined specificty of monoclonal anti-glycophorin antibodies makes them more precise tools in serological investigation and identification of normal and variant antigens. Last but not least, elucidation of antigenic properties of glycophorins is important for identification and characterization of human anti-glycophorin antibodies, which in some cases create medical problems at transfusion or pregnancy.

Subtle differences in glycosylation of blood group M and N type glycophorin A detected with anti-Tn lectins and confirmed by chemical analysis.

A higher content of Tn and sialyl-Tn receptors in glycophorin A of blood group N than in that of blood group M was suggested by reactions with anti-Tn lectins. Analysis of beta-elimination products of two blood group M and two blood group N preparations by gas liquid chromatography-mass spectrometry showed that GalNAc-ol was detectable in minor amounts in all analyzed samples and its content was higher in the products obtained from desialylated antigens. Moreover, the content of GalNAc-ol detected in blood group N samples was almost twice as high as in respective blood group M samples. Since blood group M and N antigens differ in two amino-acid residues, our results support the existence of sequence-dependent differences in efficiency of substitution of glycophorin GalNAc-Ser/Thr residues with galactose.

The differences in significance of alpha 2,3Gal-linked and alpha 2,6GalNAc-linked sialic acid residues in blood group M- and N-related epitopes recognized by various monoclonal antibodies.

The blood group M and N determinants of glycophorin A (GPA) contain O-linked oligosaccharide chains with alpha 2,3Gal-linked and alpha 2,6GalNAc-linked sialic acid residues which are required for the activity of most epitopes recognized by various anti-M and anti-N antibodies. In order to check whether these two types of sialic acid residues differ in their contribution to antigenic properties, the GPA-M and GPA-N preparations with monosialylated oligosaccharide chains were obtained and tested for binding of anti-M and anti-N monoclonal antibodies (MAbs). The GPAs with sialic acid residues linked to Gal (GPA2,3) were obtained by selective resialylation of asialoGPAs with alpha 2,3-sialyl-transferase. These preparations were tested by inhibition of binding of MAbs to enzyme-linked immunosorbent assay (ELISA) plates coated with the respective untreated target antigens. The GPAs with sialic acid residues linked to GalNAc (GPA2,6) were generated by treating GPAs adsorbed on ELISA plates with Newcastle disease virus (NDV) isolate (expressing sialidase specific for alpha 2,3Gal linkage), which was followed by testing the binding of MAbs to NDV-treated antigens. Different patterns of activity were obtained among 14 MAbs specific for sialic acid-dependent epitopes (eight anti-M and six anti-N). The results indicated that at least half of the MAbs showed distinct requirements for the presence of only one of two kinds of sialic acid residues (Gal or GalNAc linked) in the epitope. Only four MAbs (two anti-M and two anti-N) did not react with any of the 'monosialylated' forms of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)