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

Abstract

Human fl-glucuronidase (fl-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 has indicated that the enzyme exhibits charge heterogeneity and that "highuptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than, slowly internalized forms. Here we present two 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 first 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. iD-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 K is estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10-6 M; mannan from wild-type S. cerevisiae, 3 X 10-5 M; D-mannose 6-phosphate, 6 X 10-5 M; L-fucose, 4 X 10-2 M; and D-mannose, 6 X 10-2 M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet fl-glucuronidase abolished its "high-ptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.Pinocytosis of lysosomal enzymes, identified by Neufeld and coworkers (1) as uptake of corrective factors by enzyme-deficient fibroblasts, displays the selectivity and saturability expected for a receptor-mediated process (2). Several investigators (3-5) have studied kinetic aspects of this process with enzymes from different sources. Hickman and Neufeld (6) suggested that many lysosomal hydrolases have similar components that are essential for their recognition and uptake by human fibroblasts. This suggestion was based on the observation that fibroblasts from patients homozygous for a single gene mutation (I-cell disease) secrete several hydrolases which are not specifically pinocytosed (6). The finding that periodate treatment of fihexosaminidase secreted by normal fibroblasts destroyed its uptake activity led to the suggestion that the "recognition component" on the enzyme contains carbohydrate (7).We have utilized fl-glucuronidase (f3-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) to study this pinocytosis process. Previously published studies have shown (i) that human f,-glucuronidase from all tissue sources investigated exhibits charge heterogeneity (8); (ii) that high-uptake forms, which are specifically pinocytosed by fibroblasts, are more acidic than the low-uptake, or poorly ...

Mechanisms for the incorporation of proteins in membranes and organelles.

“…Secretion of precursor forms can also be induced by treatment of normal cells with the lysosomotropic drug chloroquine or with NH4C1, which alter the lysosomal pH. All these results suggest that the posttranslational modification of lysosomal hydrolase precursors by addition of phosphomannosyl residues to the oligosaccharide core, a process that is likely to occur in the Golgi apparatus (226), is necessary to divert the lysosomal enzymes from the secretory pathway and direct them to their destination (105) . This is in accordance with a model in which, in the absence of retention signals, polypeptides discharged into the ER lumen are secreted from the cell, without the need of specific signals directing the protein along the secretory route.…”

Exocytosis of pinocytic contents by Chinese hamster ovary cells.

“…2). Third, incubation of cells with HRP in the presence of saccharides known to inhibit receptor-mediated pinocytosis (8,16,20) had no effect on the uptake of HRP, a glycoprotein (Table 1) . Fourth, no cell surface binding of HRP was detected by electron microscopic cytochemistry after exposure of cells to peroxidase at either 4°C or 37°C (Fig.…”

“…The extracellular appearance of the enzyme was not due to cell lysis. Previous investigators (22) 8 Distribution of HRP activity in sucrose gradients . CHO cells were exposed to 1 mg/ml HRP for (a) 10 min or (b) 10 min followed by a 2-h chase.…”

Effects of bone marrow transplantation on the cardiovascular abnormalities in canine mucopolysaccharidosis VII

“…Secreted enzyme can ultimately reach a lysosome in another cell because the receptor is also present in the plasma membrane of most cells. [9][10][11] Thus, secreted enzymes that connect with this receptor can be internalized and transferred to a lysosome. This 'cross correction' pathway provides the rationale for BMT therapy for lysosomal storage diseases.…”