Identification of Mannose 6-phosphate in Glycoproteins that Inhibit the Assimilation of β -galactosidase by Fibroblasts

• Published in: Proceedings of the National Academy of Sciences - PNAS 1979-09, Vol.76 (9), p.4235-4239
• Main Authors: Distler, Jack, Hieber, Virginia, Sahagian, Gary, Schmickel, Roy, Jourdian, George W.
• Format: Article
• Language: English
• Subjects: beta-Galactosidase - metabolism; Biological Sciences: Biochemistry; Carbon; Carrier Proteins - metabolism; Electrophoresis; Enzymes; Fibroblasts; Fibroblasts - metabolism; Galactosidases - metabolism; Glycopeptides; Glycoproteins; Glycoproteins - analysis; Glycoproteins - metabolism; Glycoproteins - pharmacology; Hexosephosphates - pharmacology; Humans; Mannosephosphates - analysis; Mannosephosphates - pharmacology; Oligosaccharides; Peptide Fragments - pharmacology; Phosphatases; Phosphates; Receptors, Drug - metabolism; Structure-Activity Relationship; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.76.9.4235

Bovine testicular β -galactosidase (β -D-galactoside galactohydrolase, EC 3.2.1.23) is rapidly and selectively assimilated by human skin fibroblasts. The assimilation of the enzyme is strongly inhibited by mannose 6-phosphate and by a glycoprotein fraction isolated from bovine testes (glycoprotein inhibitors). These results suggest that β -galactosidase and the glycoprotein inhibitors have a common recognition marker that contains mannose 6-phosphate. The presence of mannose phosphate in the glycoprotein inhibitors was demonstrated by acid hydrolysis of the glycoproteins to liberate mannose phosphate followed by reduction with NaB3H4to give [3H]mannitol phosphate. The3H-labeled compound was identified by paper electrophoresis and by the release of [3H]mannitol on treatment with phosphatase. The [3H]mannitol phosphate was oxidized with periodate and the resulting phosphorylated fragment, on reduction with NaB3H4, yielded [3H]ethylene glycol phosphate, indicating substitution of phosphate on carbon 6 of mannitol. Mannose 6-phosphate was also found in a major carbohydrate-containing fraction of peptides produced from the glycoprotein inhibitors by trypsin digestion. It was estimated that about 2% of the mannose residues were present as mannose 6-phosphate. Phosphorylated oligosaccharides were also identified in hydrolysates of the glycoprotein inhibitors. One, a disaccharide, was identified as α -(mannosyl-6-phosphate)-(1 → 2)-mannose. These observations suggest that the recognition marker of β -galactosidase contains α 1,2-linked mannose 6-phosphate; terminal α 1,2-linked mannose residues are known to occur in the high-mannose type oligosaccharides present on β -galactosidase.