Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases

Abstract

A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting A_pae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars.

Figure 1. Amino acid sequence comparison between human A/B/cis-AB transferases, mouse cis-AB transferase, and human and mouse FS proteins.

Amino acid sequences of the coding regions of human A, B, and cis-AB transferases encoded by A101 (H_ABO-A), B101 (H_ABO-B), and cis-AB01 (H_ABO-AB) alleles of ABO gene, mouse cis-AB transferase encoded by mouse ABO gene (M_ABO-AB), human FS protein encoded by Forssman-negative GBGT1 gene (H_GBGT1) from ordinary individuals and that of Forssman-positive GBGT1 gene (H_GBGT1+) from rare A_pae individuals, and mouse FS encoded by GBGT1 gene (M_GBGT1) were aligned and are shown. When amino acids are conserved in all the proteins, they are marked above with an equal symbol (=). 116 out of 274 amino acids (42.3%) in the last 2 coding exons are conserved in all those proteins. When amino acids of mouse cis-AB transferase are identical to all the other human A/B/cis-AB transferases, they are marked above with a plus symbol (+). Similarly, when amino acids are conserved among FS proteins, they are marked below with an asterisk (*). The seven amino acids that are shared between mouse cis-AB transferase and all FS proteins are highlighted. Crucial amino acids corresponding to codons 176, 235, 266, and 268 of human A/B/cis-AB transferases, as well as codon 296 of FS proteins are indicated in bold type. Murine cis-AB transferase has the same tripeptide sequence of GlyGlyAla(GGA) as that of FS proteins.

Figure 2. Immunostaining of COS1 and COS1(B3GALNT1) cells using anti-Forssman antibody after infection with viral constructs expressing human FS proteins with and without amino acid substitutions.

Results of anti-Forssman antibody immunostaining after viral infection are compared between COS1 and COS1(B3GALNT1) cells. Cells were imaged using a Leica DMI6000B microscope equipped with a 20x/0.30 PH1 objective lens. One representative optical field was selected from individual experiments, and the cell pictures were taken in grey scale at the same magnification using monochrome DFC420 camera and Application Suite V3 software. All pictures are shown in 2 different magnifications. The right pictures correspond to the right bottom quarter of the left pictures at a higher magnification (2x). Figures were created using Adobe Photoshop software. The left panel shows the results using COS1 cells, and the right panel shows those using COS1(B3GALNT1) cells. The viral expression constructs examined are original human GBGT1 gene (H_GBGT1), human GBGT1 with Arg296Gln substitution (H_GBGT1-R296Q), human GBGT1 with Ser230Gly and Arg296Gln substitutions (H_GBGT1-S230G&R296Q), and human GBGT1 with Ser230Gly substitution (H_GBGT1-S230G) from top to bottom. Because the cells infected with the viral vectors were FACS-selected for their expression of coupled BFP and GFP, all the cells in a single experiment exhibited similar immunostaining results; positive or negative, depending on the constructs. Forssman antigen positivity is characterized not only by darker grey but also by cell surface DAB pigmentation.

Figure 3. Forssman antigen expression of COS1(B3GALNT1) cells transfected with the original and amino acid substitution constructs of mouse GBGT1 gene, mouse ABO-AB gene, and human ABO-A gene.

Results of anti-Forssman antibody immunostaining of COS1(B3GALNT1) cells after DNA transfection are shown. Cells were imaged in color using a Leica DMIL LED microscope equipped with D-LUX3 camera. All images were taken at the same magnification using a 10x/0.25 PH1 objective. Figures were created using Adobe Photoshop software. The eukaryotic expression constructs prepared in the pSG5 plasmid vector were used. The results are: mouse original GBGT1 gene (M_GBGT1) having GlyGlyAla tripeptide and its in vitro mutagenized amino acid substitution constructs with either LeuGlyGly or MetGlyAla tripeptide in the top row, mouse original ABO-AB gene (M_ABO-AB) with GlyGlyAla tripeptide and its substitution constructs having LeuGlyGly or MetGlyAla in the middle row, and human A allele of ABO gene (H_ABO-A) with LeuGlyGly tripeptide and its substitution constructs having GlyGlyAla or MetGlyAla tripeptide in the bottom row. Under the experimental conditions used, cells became confluent and formed a monolayer. Strong positive staining with anti-Forssman antibody was observed by DNA transfection of original M_GBGT1 and M_ABO-AB expression constructs. Additionally, COS1(B3GALNT1) cells transfected with H_ABO-A(GGA), the H_ABO-A construct with GlyGlyAla tripeptide sequence, exhibited some positive staining. It should be noted that not all cells were stained positive by DNA transfection as opposed to the viral infection-FACS sorting results shown in Fig. 2. Bluish background color was due to DAB and nickel ions in color development solution.