title: Bovine beta 1----4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains. In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II membrane-bound glycoproteins.
abstract: We have used S1 and primer extension analysis to demonstrate that the gene for bovine beta 1----4-galactosyltransferase specifies two sets of mRNA transcripts of different lengths. The longer mRNA transcripts initiate upstream of two in-frame ATG codons and encode a protein of 402 amino acids (long form). The shorter mRNA transcripts initiate between the two in-frame ATG codons and encode a protein of 389 amino acids (short form). These two related forms of beta 1----4-galactosyltransferase have an identical large (358 amino acids), potentially glycosylated, COOH-terminal catalytic domain, and an identical single transmembrane domain. The only difference in primary structure between the two forms is that the long form contains an NH2-terminal extension of 13 amino acids. Thus, bovine beta 1----4-galactosyltransferase fits the pattern established for murine beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. L., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428). Inspection of the NH2-terminal domain suggests that the long form of the bovine enzyme, like its murine counterpart, has a functional cleavable signal sequence which would dictate that the two forms of the membrane-bound enzyme are oriented in opposite directions. We have tested this hypothesis by in vitro translation in the absence or presence of dog pancreas microsomes. In vitro translation of RNA transcripts for the long and short form of beta 1----4-galactosyltransferase in the absence of microsomes results in the synthesis of polypeptides with apparent Mr of 44,500 and 43,000, respectively. In vitro translation of each transcript in the presence of microsomes results in the synthesis of two glycosylated, endoglycosidase H-sensitive proteins with apparent Mr of 47,500 and 46,000. These experiments and additional protease protection experiments demonstrate that the COOH-terminal domain of both the short and the long form of bovine beta 1----4-galactosyltransferase are translocated into the microsomal lumen. By extrapolation, both forms of the enzyme are oriented in vivo as Type II membrane-bound glycoproteins.
authors.affiliation: Cell Structure and Function Laboratory, Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: R N
authors.initials: RN
authors.lastname: Russo
authors.forename: N L
authors.initials: NL
authors.lastname: Shaper
authors.forename: J H
authors.initials: JH
authors.lastname: Shaper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D015723:Gene Library; D005796:Genes; D005822:Genetic Vectors; D007788:Lactose Synthase / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics*; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic
chemical_list: D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase; D015719:Single-Strand Specific DNA and RNA Endonucleases