Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Abstract

Human platelet .beta.-glucuronidase (.beta.-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work indicated that the enzyme exhibits charge heterogeneity and that high-uptake forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than slowly internalized forms. The present study presents 2 lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The 1st line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. D-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1-phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent Ki estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnn1, 3 .times. 10-6 M; mannan from wild-type S. cerevisiae, 3 .times. 10-5 M; D-mannose 6-phosphate, 6 .times. 10-5 M; L-fucose, 4 .times. 10-2 M; and D-mannose, 6 .times. 10-2 M. The 2nd line of evidence comes from the observation that alkaline phosphatase, [EC 3.1.3.1] treatment of human platelet .beta.-glucuronidase abolished its high-uptake activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.