title: alpha-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.
abstract: Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Sleat
authors.forename: S R
authors.initials: SR
authors.lastname: Kraus
authors.forename: I
authors.initials: I
authors.lastname: Sohar
authors.forename: H
authors.initials: H
authors.lastname: Lackland
authors.forename: P
authors.initials: P
authors.lastname: Lobel
mesh_terms: D000135:Acid Phosphatase / Q000652:urine; D000595:Amino Acid Sequence; D020789:Cathepsin C; D002846:Chromatography, Affinity; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000652:urine; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine*; D006026:Glycoside Hydrolases / Q000652:urine; D006801:Humans; D008360:Mannosephosphates / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013429:Sulfatases / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine*; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine*
chemical_list: D006023:Glycoproteins; D008360:Mannosephosphates; D010446:Peptide Fragments; C027693:mannose-6-phosphate; D000135:Acid Phosphatase; D013429:Sulfatases; C018719:N-acetylglucosamine-6-sulfatase; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C