An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures.
A GDP-fucose:GM1 alpha1-->2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly expressed in many rat hepatoma cell lines, including rat hepatoma H35 cells. Enzymatic properties and acceptor specificity of native rat hepatoma H35 cell alpha1-->2FucT, expressed recombinant full-length H35 cell alpha1-->2FucT, and a truncated form missing the first 27 amino acid residues from the N-terminus, comprising the cytoplasmic and transmembrane domains of the enzyme, were studied. The results indicate that the recombinant full-length enzyme has a specific activity over 80-fold higher than the truncated enzyme. Both the native and recombinant full-length enzymes display significant activity in the absence of detergent or phospholipid and optimal activity in the presence of Triton CF-54 detergent. The truncated enzyme is optimally activated by CHAPSO, showing little activity in its absence. These findings are in agreement with previous studies demonstrating a requirement of a lipidic environment for optimal activity with this enzyme and suggest that the N-terminal transmembrane domain is important either in the maintenance of an active conformation or in allowing efficient interaction with acceptor glycolipids. Both the full-length and truncated enzymes transfer fucose not only to GM1 and asialo-GM1 (Gg4) but also to galactosyl globoside (Gb5) as well. Weak or undetectable transfer to lacto- and neolacto-series acceptors was observed, demonstrating a strong preference for terminal Galbeta1-->3GalNAc- structures. The structures of two reaction products generated by expressed recombinant full-length alpha1-->2FucT, which are known to be important tumor-associated antigens (fucosyl-GM1 and fucosyl-Gb5), were unambiguously confirmed by 1H-NMR spectral analysis.
Sherwood
A L
AL
Department of Molecular Medicine, Northwest Hospital, 21720 23rd Drive SE, Suite 101, Bothell, Washington 98021, USA. asherwoo@mmnwh.org
Stroud
M R
MR
Levery
S B
SB
Holmes
E H
EH
D000595:Amino Acid Sequence; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D002240:Carbohydrate Sequence; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D055746:Stage-Specific Embryonic Antigens; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D015295:Antigens, Tumor-Associated, Carbohydrate; D003902:Detergents; D005647:Fucosyltransferases; D005677:G(M1) Ganglioside; D006028:Glycosphingolipids; D010446:Peptide Fragments; D010743:Phospholipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D055746:Stage-Specific Embryonic Antigens; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C052924:stage-specific embryonic antigen-3; C016467:Thomsen-Friedenreich antigen; C037650:GDPfucose-GM1 ganglioside alpha-fucosyltransferase