title: Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins.
abstract: Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.
authors.affiliation: Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, 08854, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Sleat
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Wang
authors.forename: Istvan
authors.initials: I
authors.lastname: Sohar
authors.forename: Henry
authors.initials: H
authors.lastname: Lackland
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zheng
authors.forename: Peter
authors.initials: P
authors.lastname: Lobel
mesh_terms: D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000378:metabolism; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D008360:Mannosephosphates; D011506:Proteins; D020543:Proteome; C018589:lysosomal proteins; C027693:mannose-6-phosphate