Peptide mapping of the subunits and deglycosylated polypeptides of human liver alpha-L-fucosidase.
The subunits of human liver alpha-L-fucosidase have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, excised, and subjected to peptide mapping after CNBr cleavage or trypsin digestion. The CNBr peptide maps of the glycosylated 56- and 51-kDa subunits were similar except that the larger subunit had several peptides with M(r)s shifted higher than those apparent for the smaller subunit. These M(r) differences were almost completely eliminated when CNBr peptide mapping was performed on the deglycosylated 48- and 45-kDa polypeptides, suggesting that the M(r) differences were due to carbohydrate differences. Minor differences not related to glycosylation were found in the CNBr peptide maps for the 48- and 45-kDa polypeptides including the presence of small amounts of three peptides in the larger polypeptide not found in the smaller polypeptide. Sequence analysis suggested that both the 48- and the 45-kDa polypeptides were blocked at their amino-termini but analysis of the largest CNBr peptide from each polypeptide indicated an identical 13-amino-acid sequence corresponding to residues 6 through 18 from the cDNA-deduced sequence of mature alpha-L-fucosidase. Tryptic peptide mapping indicated very similar HPLC peptide profiles for the deglycosylated 48- and 45-kDa polypeptides except for the presence of small amounts of six peaks present in the larger polypeptide which were not detected in the smaller polypeptide. The overall results provide the first evidence that the polypeptides of human liver fucosidase are very similar and probably encoded by the same gene. However, minor differences in the polypeptides exist, possibly due to normal allelic variation, alternative splicing, proteolytic processing, and/or posttranslational modifications other than those due to glycosylation.
Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015.
F
F
Shoarinejad
J
J
Zhu
S B
SB
Bazel
J A
JA
Alhadeff
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D006020:Glycopeptides / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000737:chemistry; D017421:Sequence Analysis; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000378:metabolism
D006020:Glycopeptides; D010455:Peptides; D005644:alpha-L-Fucosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003488:Cyanogen Bromide