The structures of asparagine-linked oligosaccharides of rat liver cathepsin L reflect the substrate specificity of lysosomal alpha-mannosidase.
We have studied the structures of asparagine-linked oligosaccharides of cathepsin L purified from rat liver in detail. The oligosaccharides released from rat liver cathepsin L on glycopeptidase-F treatment were tagged with 2-aminopyridine at their reducing ends. The pyridylamino (PA) derivatives were separated into seven fractions according to molecular size by normal-phase HPLC. The structure of each oligosaccharide thus isolated was analyzed by reversed-phase HPLC and characterized by ion-spray mass spectrometry and high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our results indicate that the asparagine-linked oligosaccharides of rat liver cathepsin L are of the oligomannose type, having two to six mannose residues. Among them, the five major ones are Manalpha1-6Manbeta1-4-GlcNAcbeta1-4GlcNAc, Manalpha1 -6Manalpha1-6Manbeta1-4GIcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)-Manalpha1-6Manbeta1- 4GlcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-++ +2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4Glc-NAc. Their structures are shown to be products of Man6GlcNAc2 hydrolysis with lysosomal alpha-mannosidase.
Towatari
T
T
Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Japan. towatari@ier.tokushima-u.ac.jp
Miyamura
T
T
Kondo
A
A
Kato
I
I
Inoue
M
M
Yano
M
M
Kido
H
H
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001692:Biological Transport; D002240:Carbohydrate Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry*; D002451:Cell Compartmentation; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D006023:Glycoproteins; D009844:Oligosaccharides; D001216:Asparagine; D008361:Mannosidases; D043323:alpha-Mannosidase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat