Cloning and Expression of the Histo-blood Group PkUDP-galactose:Galβ1–4Glcβ1-Cer α1,4-Galactosyltransferase

Abstract

The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P 1 , P, and P k glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Gal␤1-R 4-␣-galactosyltransferases catalyze the syntheses of the structurally related P 1 and P k antigens. The P 1 polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an ␣4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated ␣4Gal-T1. Expression of full coding constructs of ␣4Gal-T1 in insect cells revealed it encoded P k but not P 1 synthase activity. Northern analysis showed expression of the transcript correlating with P k synthase activity and antigen expression in human B cell lines. Transfection of P k -negative Namalwa cells with ␣4Gal-T1 resulted in strong P k expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that ␣4Gal-T1 represented the P k gene. Sequence analysis of the coding region of ␣4Gal-T1 in P 1 ؉/؊ individuals did not reveal polymorphisms correlating with P 1 P 2 typing.The P histo-blood group system is the last of the known carbohydrate defined blood group systems for which the molecular genetic basis has not yet been clarified. The P blood group system involves two major blood group phenotypes, P 1 ϩ and P 1 Ϫ, with approximate frequencies of 80% and 20%, respectively (1, 2). P 1 Ϫ individuals normally express the P antigen (P 1 Ϫ is designated P 2 when P antigen expression is demonstrated), but the rare P k phenotype lacks the P antigen, while the rare p phenotype lack both P and P k antigens (for reviews, see Refs. 3-7). The P 1 ϩ phenotype is defined by expression of the neolacto-series glycosphingolipid P 1 (for structures, see Table I) (8). In contrast, the P, P k , and p antigens constitute intermediate steps in biosynthesis of globo-series glycolipids and give rise to P 1 k , P 2 k , and p phenotypes (9). Although the rare P k phenotype shows the same frequency of P 1 antigen expression as individuals expressing the P antigen, the p phenotype is always associated with lack of P 1 antigen expression. Extensive studies of the chemistry, biosynthesis, and genetics of the P blood group system identified the antigens as being exclusively found on glycolipids, with the blood group specificity being synthesized by at least two distinct glycosyltransferase activities; UDP-galactose:␤-D-galactosyl-␤1-R 4-␣-Dgalactosyltransferase (␣4Gal-T) 1 activity(ies) for P k and P 1 syntheses and UDP-GalNAc:Gb 3 3-␤-N-acetylgalactosaminyltransferase activity (EC 2.4.1.79) for P synthesis (for reviews, see Refs. 6 and 7). At least two independent gene loci, P and P 1 P k , are involved in defining these antigens. The P blood group-associated LKE antigen, shown to be the extended sialylated Gal-globoside structure (10), may involve polymorphism in an ␣2,3-sialyltransferase activity.A long-standing controversy has been whether a single...

Molecular Cloning of Pigeon UDP-galactose:β-d-Galactoside α1,4-Galactosyltransferase and UDP-galactose:β-d-Galactoside β1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Galα1–4Galβ1–4Galβ1–4GlcNAc Sequence

“…There are three sequences in zebra finch and one sequence in chicken, and these sequences were annotated "similar to ␣1,4-galactosyltransferase." The BLAST analysis also revealed human (16,17), mouse (18), and rat (19) ␣4GalT(Gal)s as being homologous proteins, as well as some other predicted proteins from mammals that were annotated similar to ␣1,4-galactosyltransferase. The pigeon ␣4GalT(Gal) also has some homology with the human ␣1,4-N-acetylglucosaminyltransferase (␣1,4-N-acetylglucosaminyltransferase, 36.3% identity; accession number AAD48406) and putative ␣1,4-N-acetylglucosaminyltransferase from chicken (34.4%, accession number XP_426692) and zebra finch (31.5%, accession number XP_002189476).…”

“…Because the Gal␣1-4Gal sequence is rarely attached to glycoproteins in mammals, the putative ␣4GalT(Gal) in pigeon is likely to have distinct acceptor substrate specificities from those of mammals. Although mammalian ␣4GalT(Gal)s, also known as Gb3 synthases, have been cloned from human (16,17), mouse (18), and rat (19), they utilize lactosylceramide (Gal␤1-4Glc-Cer) as the preferred acceptor substrate to produce Gb3. Only when the expression level of the enzyme is sufficiently high, human Gb3 synthase can also synthesize P 1 antigen using lacto-N-neotetraosylceramide (Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-Cer) as a substrate (20).…”

The Blood Group P1 Synthase Gene Is Identical to the Gb3/CD77 Synthase Gene

“…On the other hand, the occurrence of the p phenotype is very rare in all nationalities (18,19), suggesting that the genetic basis of p and P2 phenotypes is not directly linked. Actually, no relevant polymorphic sequences in the coding region of the Gb3/CD77 synthase gene could be detected between P1 and P2 individuals, and no P1 synthase activity could be found in cell lysates transfected with a Gb3/CD77 synthase expression vector (8,9). Consequently, it appears difficult to accept that P1 synthase and Gb3/CD77 synthase are identical.…”

“…A stable transfectant of L cells (L-VTR) was established as described previously (9) and maintained in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum and G418 (Invitrogen) (300 g/ml).…”

“…Lack of Gb3 synthase results in p phenotype expressing neither Gb3 nor P (Gb4), because P is generated from Gb3 with Gb4 synthase (12). In fact, multiple mutations in the Gb3/CD77 synthase gene leading to functional loss of the enzyme activity were identified in the individuals with p phenotype (9,13). On the other hand, P1/P2 (P1 negative) is the last glycolipid antigen system for which the genetic mechanisms has not yet been clarified, because the P1 synthase gene has not been isolated to date.…”