Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli.
Glycogenin is a self-glucosylating protein involved in the initiation reactions of glycogen synthesis. Initiation occurs in two stages, requiring first the covalent attachment of a glucose residue to Tyr-194 of glycogenin and then elongation to form an oligosaccharide chain. The latter reaction is known to be catalyzed by glycogenin itself. The glycogenin sequence determined from the protein by Campbell and Cohen (Campbell, D. G., and Cohen, P. (1989) Eur. J. Biochem. 185, 119-125) was used to design oligonucleotide probes to screen a rabbit muscle lambda gt11 library. A cDNA was isolated that predicted an amino acid sequence identical to that of Campbell and Cohen, except that Cys residues replaced Ser-88 and Leu-97. Northern analysis indicated a strongly hybridizing message of 1.8 kilobases, present in most tissues including skeletal muscle, but much weaker in kidney and scarcely detectable in liver. A much weaker 3-kilobase message was also detected in muscle. Polymerase chain reaction was used to isolate DNA fragments encoding a portion of glycogenin from rat and cow. The sequence of this segment was > 90% identical at the amino acid level across the three species, indicating that glycogenin is a highly conserved protein. Using the pET-8c vector, the glycogenin protein was expressed in Escherichia coli. Incubation of the recombinant glycogenin with UDP-[14C]glucose and Mn2+ resulted in labeling of the glycogenin protein, indicating that the recombinant glycogenin was enzymatically active and capable of self-glucosylation. Furthermore, after incubation with UDP-glucose, the recombinant glycogenin could serve as a substrate for glycogen synthase, leading to the production of high M(r) polysaccharide. Therefore, production of functional glycogenin did not require the intervention of any other mammalian protein.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005964:Glucosyltransferases; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000235:genetics; D009132:Muscles / Q000378:metabolism*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D006023:Glycoproteins; D009124:Muscle Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C051887:glycogenin; D012313:RNA; D005964:Glucosyltransferases