Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme.
The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Henion
T R
TR
Department of Microbiology and Immunology, Medical College of Pennsylvania, Philadelphia 19129.
Macher
B A
BA
Anaraki
F
F
Galili
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002144:Callithrix; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002427:Cebidae; D002520:Cercopithecidae; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D015186:Hominidae; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D011443:Strepsirhini; D014162:Transfection
D009844:Oligosaccharides; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C037959:beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase