Fibroblast receptor for lysosomal enzymes mediates pinocytosis of multivalent phosphomannan fragment

Abstract

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human β-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human β-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human β-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.

Investigations on β-Glucuronidase-Positive Erythrocytic Inclusions

“…The uptake of exogenous [3-glucuronidase by erythrocytes under certain pathological circum stances may resemble that by fibroblasts [17][18][19][20][21][22], and we have found an elevated level of [3-glucuronidase [23] in the blood plasma of three patients with these inclusions. Studies on the formation of [3-glucuroni dase-positive inclusions in erythrocytes are in prog ress.…”

The Phosphomannosyl Recognition System for Intracellular and Intercellular Transport of Lysosomal Enzymes

“…Mild acid hydrolysis of native H. holstii phosphomannan yields two principal products [34, 351, a monovalent pentamannosylmonosphosphate, (Man),-P, and a larger molecular weight polyphosphomonoester (PPME) fragment, which are quite different in their potency as pinocytosis inhibitors [30]. The large PPME, which can be regarded as multivalent with many exposed 6-phosphomannose groups, is 100-fold more potent as an enzyme pinocytosis inhibitor per mole of phosphate (100,000-fold more potent per molecule) than is (M~II)~-P [32]. Moreover, the rate and kinetics of (Man),-P uptake are no greater than can be explained solely by nonspecific, fluid-phase endocytosis.…”

Identification of a variant of mucolipidosis III (pseudo-Hurler polydystrophy): a catalytically active N-acetylglucosaminylphosphotransferase that fails to phosphorylate lysosomal enzymes.

“…This variant form of pseudo Hurler polydystrophy demonstrates the biological importance of this recognition mechanism in the generation of the phosphomannosyl marker. Acid hydrolases are a heterogeneous group of proteins that acquire a common phosphomannosyl recognition marker which is responsible for targeting them to their common destination, the lysosomes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). This posttranslational modification of the high-mannose-type oligosaccharides of lysosomal enzymes is generated by the sequential action of two enzymes: UDP-Nacetylglucosamine:lysosomal enzyme N-acetylglucosamine-lphosphotransferase [referred to herein as N-acetylglucosaminylphosphotransferase (GlcNAcPlase)] and a-N-acetylglucosaminyl phosphodiesterase.…”