Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family

Abstract

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the “Dombrock” blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5′-diphosphate (ADP)–ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Doa versus Dob antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Two new molecular bases for the Dombrock null phenotype

“…The cloning of the DO gene (Gubin et al, 2000) has made it possible to determine the molecular basis of antigens in the Dombrock blood group system (Rios et al, 2001a(Rios et al, , 2002. The molecular basis for two types of Gy(a-) have also been reported: one type is caused by a mutation of a to g in the acceptor splice site of intron 2 (IVS1-2 a > g) (Rios et al, 2001b) and the other by a deletion of nucleotides 343-350 in exon 2 (Bailly et al, 2001).…”

“…RNA was extracted with Trizol reagent (Gibco, Gaithersburg, MD, USA) from GY4 and first-strand cDNA synthesized using SUPERSCRIPT TM Preamplification System for First Strand cDNA Synthesis Kit (Gibco), with oligo-dT as the primer. The cDNA was amplified using the AdvantageÒ cDNA PCR kit (Clontech, Palo Alto, CA, USA), using the conditions and primers described previously (sense 5¢-ATGGGTCCATTGATCAACAGATGCAAGA-3¢ located in the non-translated region of exon 1 and antisense 5¢-TTATACTCTGCTTTTGGAAAAGATGATGA-3¢ located in the non-translated region of exon 3) (Gubin et al, 2000). This primer pair is expected to generate a PCR product of 945 bp from normal DO or of 236 bp if exon 2 were outspliced.…”

“…Sequences of the PCR product from GY4 showed 378T, 624C and 793G, which are the nucleotides associated with the DOB allele (Gubin et al, 2000;Rios et al, 2001a). The sequence for GY4 also had a homozygous t to c mutation in the 5¢ donor splice site of intron 1 (IVS1 + 2t > c).…”

“…Three additional antigens of high incidence, Gregory (Gy a ; ISBT 014003), Holley (Hy; ISBT 014004) and Joseph (Jo a ; ISBT 014005), are also carried on the Dombrock glycoprotein, which is a glycosylphosphatidylinositol (GPI)-linked protein on red blood cells (RBCs) (Telen et al, 1990;Spring & Reid, 1991;Spring et al, 1994;Banks et al, 1995). The DOA and DOB alleles are associated with three nucleotide changes, respectively, 378 C > T, 624 T > C and 793 A > G in the coding region of DO (GenBank Accession number XM_017877) (Gubin et al, 2000;Rios et al, 2001a). Two of the three polymorphisms (nucleotides 378 and 624) are silent at the amino acid level and correspond to 126 tyrosine and 208 leucine.…”

The Dombrock blood group system: a review

“…11,19 It has several potential N-linked glycosylation sites and four or five cysteine residues in the membrane bound protein. 23 The susceptibility of Do antigens to sulfydryl compounds suggests that the tertiary conformation of the glycoprotein is dependent on disulfide bonds.…”