ISSN:
0730-2312
Keywords:
phosphomannosyl receptor
;
pinocytosis
;
fibroblast secretions
;
glycopeptides
;
acid hydrolases
;
lysosomotropic amines
;
β-hexosaminidase B
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 × 10-6 M) and for the uptake of secretion acid hydrolases (Ki of 2.2 × 10-6 M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 × 10-5 M) compares closely to that previously determined for the pinocytosis of intact “high-uptake” acid hydrolases (3-6 × 10-5 M).“High-uptake” fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand. The pinocytosis rates (% of added ligand internalized/mg protein/hr) of the “high-uptake” fraction of both intact acid hydrolase (12%/mg/hr) and glycopeptide (6%/mg/hr) differed by only twofold. The apparent Kuprake for both ligands was of the same order of magnitude. The similarity in the kinetics of pinocytosis of the secreted acid hydrolases and of the phosphomannose-bearing glycopeptides prepared from them suggests that the structural information which confers high-affinity binding to the phosphomannosyl receptor is contained in the glycopeptide units themselves. No additional information from the intact protein backbone appears essential for phosphomannosyl receptor-mediated pinocytosis.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240220202
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