title: Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation.
abstract: Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.
authors.affiliation: Amgen Inc., Thousand Oaks, CA.
authors.forename: H S
authors.initials: HS
authors.lastname: Lu
authors.forename: F K
authors.initials: FK
authors.lastname: Lin
authors.forename: L
authors.initials: L
authors.lastname: Chao
authors.forename: J
authors.initials: J
authors.lastname: Chao
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007610:Kallikreins / Q000302:isolation & purification / Q000652:urine*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases
chemical_list: D010446:Peptide Fragments; D010447:Peptide Hydrolases; D007610:Kallikreins