α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine

David E. Sleat, Stephen R. Kraus, Istvan Sohar, Henry Lackland, Peter Lobel

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 α-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 β-mannosidase (82%), hexosaminidase (27%) and α-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.

Original languageEnglish (US)
Pages (from-to)33-39
Number of pages7
JournalBiochemical Journal
Volume324
Issue number1
DOIs
StatePublished - May 15 1997

Fingerprint

N-acetylglucosamine-6-sulfatase
Glucosidases
Glycoproteins
Urine
Cations
Mannose
Sequence Analysis
Enzymes
Mannosidases
Cerebroside-Sulfatase
IGF Type 2 Receptor
Affinity chromatography
Hexosaminidases
Enzyme activity
Molecular mass
Lysosomes
mannose-6-phosphate
Affinity Chromatography
Polyacrylamide Gel Electrophoresis
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sleat, David E. ; Kraus, Stephen R. ; Sohar, Istvan ; Lackland, Henry ; Lobel, Peter. / α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine. In: Biochemical Journal. 1997 ; Vol. 324, No. 1. pp. 33-39.
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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 α-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 β-mannosidase (82{\%}), hexosaminidase (27{\%}) and α-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.",
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α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine. / Sleat, David E.; Kraus, Stephen R.; Sohar, Istvan; Lackland, Henry; Lobel, Peter.

In: Biochemical Journal, Vol. 324, No. 1, 15.05.1997, p. 33-39.

Research output: Contribution to journalArticle

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T1 - α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine

AU - Sleat, David E.

AU - Kraus, Stephen R.

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N2 - 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 α-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 β-mannosidase (82%), hexosaminidase (27%) and α-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.

AB - 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 α-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 β-mannosidase (82%), hexosaminidase (27%) and α-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.

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