Abstract
Histo-blood group A transferase produces A antigens and transfers GalNAc to the acceptor substrate, H structures of glycolipids and glycoproteins. B transferase transfers galactose in place of GalNAc to the same acceptor substrate to synthesize B antigens. We have previously identified four amino acid substitutions between human A and B transferases. Out of these four, substitutions at the last two positions (codons 266 and 268) were found to be crucial for the different donor nucleotide-sugar specificities between A and B transferases as analyzed by gene transfer of chimeric A-B transferase genes. In the present study, we have in vitro mutagenized codon 268 of these two transferase cDNA expression constructs (glycine and alanine in A and B transferases, respectively) and produced substitution constructs with every possible amino acid residue at this position. We examined the activity and specificity of each construct by gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs on the A transferase backbone with alanine, serine, and cysteine expressed enzymes with A and B transferase activities. Weak A activity was detected with histidine and phenylalanine constructs while weak B activity was detected with asparagine and threonine constructs. All the other amino acid substitutions at codon 268 on the A transferase backbone showed neither A nor B activity. The glycine construct on the B transferase backbone expressed both A and B transferase activities. Some substitution constructs on the B transferase backbone maintained B activity while some other substitutions abolished the activity. These results show that the side chain of the amino acid residue at 268 of the human A and B transferases is responsible for determining both activity and nucleotide-sugar donor substrate specificity and strongly suggest its direct involvement in the recognition of and binding to the sugar moiety of the nucleotide-sugars.
Highlights
Glycoproteins and glycolipids are essential components of “advertisement” in accordance with 18 U.S.C
O alleles are incapable of producing functional enzymes, and the substrates remain unchanged. In addition to these three major alleles, there are many other minor subtypes. They are rare in the population, glycosyltransferases coded by these subtypic alleles offer clues about the structural basis of the enzymatic activity of glycosyltransferases
We have recently identified a second type of O allele that is devoid of the single nucleotide deletion found in all other O
Summary
Materials—The reagents for PCR1 were purchased from PerkinElmer, and reagents for DNA sequencing were from U. Construction of Amino Acid Substitution Constructs—We employed polymerase chain reaction with degenerate oligodeoxynucleotides to introduce mutations at codon 268 of human A and B transferases (Fig. 1). The nucleotide sequence of codon 268 and its surrounding region (from nucleotide to nucleotide 885) of 381 and 259 clones of A and B transferase amino acid substitution constructs, respectively, was determined for individual clones. Further DNA sequencing was performed over the entire coding region, which was amplified by PCR (from the SstII site to the termination codon between nucleotides 470 and 1065) in order to exclude the clones with any additional unexpected mutation(s). DNA from a single representative clone (whose coding sequence was confirmed to be correct over the PCR-amplified region) from each amino acid substitution was cotransfected with DNA from pSV-b-galactosidase control vector. Activity was determined for each sample by measuring hydrolysis of o-nitrophenyl-b-D-galactopyranoside, a substrate for b-galactosidase, and the resulting value was used to normalize DNA transfection efficiency
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