The inter-locus recombinant HLA-B*4601 has high selectivity in peptide binding and functions characteristic of HLA-C.

The vast majority of new human HLA class I alleles are formed by conversions between existing alleles of the same locus. A notable exception to this rule is HLA-B*4601 formed by replacement of residues 66-76 of the alpha 1 helix of B*1501 by the homologous segment of Cw*0102. This inter-locus recombination, which brings together characteristic elements of HLA-B and HLA-C structure, is shown here to influence function dramatically. Naturally processed peptides bound by B*4601 are distinct from those of its parental allotypes B*1501 and Cw*0102 and dominated by three high abundance peptides. Such increased peptide selectivity by B*4601 is unique among HLA-A,B,C allotypes. For other aspects of function, presence of the small segment of HLA-C-derived sequence in an otherwise HLA-B framework converts B*4601 to an HLA-C-like molecule. Alloreactive cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and cellular glycosidases all recognize B*4601 as though it were an HLA-C allotype. These unusual properties are those of an allotype which has frequencies as high as 20% in south east Asian populations and is associated with predisposition to autoimmune diseases and nasopharyngeal carcinoma.

Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid.

Reaction conditions for conjugation of two fluorescent ortho-substituted aniline derivatives, 2-amino benzamide (2-AB) and 2-anthranilic acid (2-AA), to N- and O-glycans have been investigated. Conjugation conditions for attaching 2-AB and 2-AA to core-fucosylated and non-fucosylated glycans were developed using complex N-glycans radiolabeled at the nonreducing terminus with [3H]C6-galactose. Optimal conditions for each of the following reaction parameters were experimentally defined: [glycans], [2-AB] or [2-AA], solvent and acid composition, temperature and time of Schiff's base formation, nature of reductant, and temperature and time of reduction. Using the optimized reaction conditions it has been shown with several standard glycans and glycoprotein-derived glycan libraries that (i) molar labeling efficiencies are high and essentially independent of the amount of glycans; (ii) negligible (< 2 mol%) desialylation occurs during conjugation; (iii) glycan labeling is nonselective, i.e., independent of glycan structure; and (iv) insignificant fluorescent or chemical "blank" is recovered during the glycan-labeling and purification protocol. Labeling glycan pools with 2-AB or 2-AA therefore allows representative glycan profiles to be obtained and also allows relative molar quantitation of individual glycans in a pool. The 2-AB label is compatible with several chromatographic means for separation of carbohydrates including Bio Gel P4 gel permeation, high-performance anion-exchange chromatography with fluorescence detection, and a variety of HPLC procedures, as well as with mass spectrometric methods including matrix-assisted laser desorption-mass spectrometry and electrospray-mass spectrometry. The 2-AA label is particularly well-suited for electrophoretic separations by polyacrylamide gel electrophoresis. These fluorophores show high intrinsic sensitivity and thus facilitate very sensitive analysis of protein glycosylation.

Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.

Apolipoprotein[a] polymorphism has been investigated by sodium dodecyl sulfate polyacrylamide (5.37%) gel electrophoresis and immunoblotting using a standardized sample load in four ethnic groups: German, Ghanaian, Chinese, and San (Kalahari Bushmen). A total of 10 different apparent molecular weight (Mr) polymorphs, designated 1 to 10 with increasing Mr, were detected in greater than 99% of all individuals tested (German, 99%; Ghanaian, 99%; Chinese, 100%; San 100%). A null allele is therefore at most an infrequent variant in all populations. Polymorphs 6-10 were common to all four populations, while polymorphs 1-5 appeared to be relatively rare variants not universally detected in each group in the present study. The Chinese had the highest proportion of double-band phenotypes and the observed frequencies were not significantly different from those expected according to simple Mendelian inheritance, whereas the observed apo[a] phenotype distributions of the other three groups did not concur with those expected for Hardy Weinberg equilibrium. The German and Ghanaian groups displayed similar distributions of apo[a] phenotypes while the Chinese and San had significantly higher frequencies of polymorphs 9 and 10. Mean plasma Lp[a] concentrations in Ghanaians (36.2 +/- 31.5 mg/dl) were almost 2-fold greater than in Germans (18.7 +/- 23.1 mg/dl) and ca 1.65-fold greater than in either Chinese (22.9 +/- 18.3 mg/dl) or San (21.1 +/- 19.3 mg/dl). A strong inverse correlation was observed between apo[a] Mr and plasma Lp[a] concentration in Germans but this was much less pronounced in Ghanaians. While the mean plasma Lp[a] levels associated with polymorphs 1-6 were similar in both Germans (43.4 +/- 30.0 mg/dl) and Ghanaians (49.2 +/- 37.6 mg/dl), those Ghanaians with any combination of the polymorphs 9 and 10 had an almost 3-fold greater mean plasma Lp[a] level (20.6 +/- 11.3 mg/dl) than their German counterparts (7.8 +/- 5.7 mg/dl). It is therefore apparent that: 1) differences in apo[a] allele frequencies are not primarily responsible for differences in Lp[a] levels between populations; and 2) the greatest ethnic variation is observed in plasma Lp[a] concentrations associated with the high molecular weight apo[a] polymorphs.

Treatment of polycythemia vera by isovolemic large-volume erythrocytapheresis.

Excess red blood cells (RBC) in patients with polycythemia vera (PV) are usually removed by repeated phlebotomy. In order to improve the efficacy of this treatment, we used isovolemic large-volume erythrocytapheresis (EA) by a cell separator. A retrospective analysis of our experience with 69 PV patients (206 EA procedures) is reported. EA induced a rapid, well-tolerated, and long-lasting reduction of Hct, Hb, and RBC counts, as well as an immediate disappearance or reduction of clinical symptoms of PV, while tissue oxygen tension - as measured in 8 patients - increased. Hct was reduced by EA from 56.8% +/- 5.6% to 41.9% +/- 6.6%, Hb from 17.5 +/- 2.3 to 12.7 +/- 2.4 g%, RBC counts from 7.4 +/- 0.9 to 5.4 +/- 0.9 x 10(6)/mm3. The mean volume of the apherisate was 1410 +/- 418 ml, (mean Hct 79.7% +/- 9.3%), and the actual RBC volume removed 1113 +/- 367 ml. The isovolemic procedure was well tolerated and the acceptance by patients seemed to be better than with repeated phlebotomy. In 21 patients whose Hct values (Hct before and after EA 58% +/- 5.7% and 41.5% +/- 4.9%) were regularly followed after EA the mean period with Hct less than 50% after a single EA procedure was 6.1 +/- 4.1 months (median, 6); in 14 out of these 21 patients a Hct of less than 43% after EA was reached and their mean period with Hct less than 50% after EA was 7.6 +/- 4.0 months (median, 7.5). For three patients this period was 11, 13, and 15 months, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)