Soluble 14-kDa beta-galactoside-specific bovine lectin. Evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain.
The soluble beta-galactoside-specific bovine lectin of subunit 14 kDa has been expressed in vitro by transcription and then translation in a rabbit reticulocyte lysate. The protein thus expressed shows the predicted binding to lactose coupled to Sepharose. Several mutants of the 134 amino acid protein have been expressed and insight gained into (a) the polypeptide length required to form the carbohydrate recognition domain and (b) the functional importance of some of the highly conserved amino acids. The following amino acids have been deleted: 1-9, 1-23, 88-122, 88-134, 107-134, or 124-134. In addition, a frame-shift mutant has been made in which the 23 amino acids at the C-terminal end were completely changed. Among these seven mutants only mutant 1-9 shows carbohydrate binding but with congruent to 30% of the activity of the wild-type protein (as assessed by the percentage of the protein bound to lactose-Sepharose). On the other hand, carbohydrate binding is relatively well preserved (75-90%) in mutant proteins where the C-terminal octapeptide sequence of the bovine lectin has been changed to sequences that resemble those in the chick 14-kDa lectin. When the single tryptophan at position 68 is changed by point mutagenesis to phenylalanine or to a leucine residue, a weak binding activity (congruent to 20%) is retained only with the former. When either of the cysteines 2 or 60 is changed to serine, binding activity is reduced to congruent to 60%, and when both are changed, to congruent to 20% of that for the wild-type protein. The susceptibility of the lectin to oxidative inactivation is unaffected when these 2 cysteines and cysteine 130 are changed to serine individually or in tandem (cysteines 2 and 60). In a second approach we show that the natural protein isolated from bovine heart is protected from proteolysis by trypsin and V8-protease in the presence of saccharide ligand. Although further work is required to identify residues which come into contact with the carbohydrate ligand, these results indicate that almost the complete polypeptide chain is necessary for the integrity of the carbohydrate recognition domain.
Abbott
W M
WM
Glycoconjugates Section, Clinical Research Centre, Harrow, Middlesex, United Kingdom.
Feizi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D006868:Hydrolysis; D037102:Lectins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism; D014176:Protein Biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D037102:Lectins; D009844:Oligosaccharides; D001616:beta-Galactosidase