Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of β-galactosidase by fibroblasts

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Abstract
Bovine testicular .beta.-galactosidase 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 .beta.-galactosidase and the glycoprotein inhibitors have a common recognition marker that contains mannose 6-phosphate. The presence of mannose 6-phosphate in the glycoprotein inhibitors was demonstrated by acid hydrolysis of the glycoproteins to liberate mannose phosphate followed by reduction with NaB3H4 to give [3H]mannitol phosphate. The 3H-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 .apprx. 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 .alpha.-(mannosyl-6-phosphate)-(1 .fwdarw. 2)-mannose. These observations suggest that the recognition marker of .beta.-galactosidase contains .alpha.1,2-linked mannose 6-phosphate; terminal .alpha.1,2-linked mannose residues are known to occur in the high-mannose type oligosaccharides present on .beta.-galactosidase.