Expansion of the Knops blood group system and subdivision of Sl(a).

BACKGROUND: Complement receptor type 1 (CR1), which bears the Knops (Kn [KN]) blood group antigens, is involved in the rosetting of Plasmodium falciparum- infected RBCs with uninfected cells. As a first step in understanding this interaction, the molecular basis for the blood group antigens encoded by CR1 was investigated. STUDY DESIGN AND METHODS: An antibody from a white donor who exhibited an apparent anti-Sl(a) was used for population studies of several racial groups. The donor's genomic DNA was sequenced to identify the Sl(a) mutation and other mutations. RESULTS: The donor with anti-Sl(a) typed as Sl(a+) with some sera and had the CR1 genotype AA at bp 4828 (R1601). However, she was homozygous for a new mutation (GG) at bp 4855 changing amino acid 1610 from S1610 to T1610 (S1610T). This mutation occurred in heterozygous form in eight white and one Asian donor. The site is only nine amino acids from the previously described Sl(a) polymorphism and appears to produce a new conformational epitope. CONCLUSION: The antigen formerly known as Sl(a) can now be subdivided. A new terminology is proposed that recognizes both linear and conformational epitopes on the CR1 protein. At amino acid 1601, Sl 1 (Sl(a)) is represented by R, Sl 2 (Vil) is represented by glycine, and Sl 3 requires both R1601 and S1610. Sl 4 and Sl 5 are hypothetical epitopes represented by S1610 and T1610, respectively.

Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1.

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.

Blood group associations with parasites, bacteria, and viruses.

Although recent investigations into the human blood groups have proceeded mainly at the molecular level, the RBC remains an exquisite model to study the expression of various genes and their related proteins. Although DNA may be informative, it may not always give meaningful information regarding protein expression on cell surfaces, which is where binding occurs. Because of their easy accessibility, RBCs will continue to be used as a major tool in the investigation of the causative agents for disease, whether they be viral, bacterial, or parasitic in nature.

Fine specificities of murine anti-Mg monoclonal antibodies.

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.

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells.

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.

Anti-Holley detected in a primary immune response.

Anti-Holley (Hy) has been reported as an IgG antibody occurring in previously transfused or multiparous black patients. In this case anti- Hy was identified in a 16-year-old black, primigravida female admitted at 32 weeks gestation because of premature rupture of the membranes. On admission, her blood type was determined to be A2B, D-positive and an antibody screen was negative. A second antibody screen, performed 4 days later, was positive in all three cells. Anti-Hy was subsequently identified. The antibody was reactive at room temperature, 37 degrees C, and in the antiglobulin phase. IgG and IgM components of anti-Hy were demonstrated in the maternal serum, documenting a primary immune response. This resulted in serologic findings not previously described for anti-Hy. A direct antiglobulin test on the newborn red cells was negative and there was no clinical evidence of hemolytic disease of the newborn (HDN). A monocyte monolayer assay performed with maternal serum yielded negative results. Recent scientific information has resulted in the placement of Hy in the Dombrock blood group system. Alloantibodies to Dombrock system antigens have not been associated with severe HDN.

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Blood grouping using a galvanic immunoelectrode sensor.

Traditional blood grouping techniques have been performed using either direct or indirect hemagglutination or adherence methods. Most procedures are time-consuming to perform, labor intensive and, for the most part, have subjective interpretation. An immunoelectrode system using a pair of electrodes, with either monoclonal antibody or red cell membrane attached to one of the electrode surfaces, has been developed. The fluid (whole blood) to be analyzed is used as an electrical bridge between the electrodes. The analysis of the fluid sample for predetermined immunological reactions can be evaluated by controlling and measuring either the current or the voltage across the two electrodes of the pair. Tests using a printed electrical circuit card of pairs of electrodes (one of the pair coated with a reactant), or a series of electrodes (each coated with a different reactant) with one common reference electrode indicate that the test procedure is rapid (less than 60 seconds) and specific. Tests results read in one hundredth of a second intervals and read at the millivolt or microvolt levels can be electronically scanned, processed through the logic table and immediately interpreted.

An anti-EnaTS detected in the serum of an MiI homozygote.

The serum of EH reacted with all red cells (RBCs) except her own, ficin- or trypsin-treated red cells, and En(a-) red cells. This reactivity defined an anti-EnaTS specificity. The red cells of the proposita typed as M-N+S-S+, Vw+Mur-Hil-Hut-Anek-Lane-, Wr(a-b+), EnaKT+. Red cells of five relatives were Vw+ and positive with her serum. Titration studies suggest that EH is genetically an MiI homozygote and that her Vw+ relatives are MiI heterozygotes. There is no history of consanguinity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting studies have agreed with the serologic observations. A variant sialoglycoprotein of faster mobility than normal glycoprotein A, but no normal glycoprotein A, was detected on her red cells. Treatment with N-glycanase did not alter the mobility, which indicated that there was no N-glycosylation of residue 26. These findings are in agreement with the reported properties of the Mi.I-specific glycoprotein A. The relatives' Vw+ red cells showed the variant sialoglycoprotein and normal glycoprotein A. EH appears to be the first reported MiI homozygote.