Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Abstract

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl alpha-d-mannoside, alpha-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for beta-glucuronidase from human urine. The inhibition of alpha-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl alpha-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A.74, 2026-2030]. Endocytosis of ;low-uptake' forms of alpha-N-acetylglucosaminidase and alpha-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that ;low-uptake' forms are either contaminated with ;high-uptake' forms or are internalized via the same route as ;high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver beta-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

Mutational Analysis of the Binding Site Residues of the Bovine Cation-dependent Mannose 6-Phosphate Receptor

“…Although the R111A and R111K constructs were poorly secreted, similar results were obtained when these substitutions were generated in the fully glycosylated truncated CD-MPR, STOP 155 (22), which resulted in their efficient secretion (80%) by transfected COS-1 cells; no detectable binding of the R111A/STOP 155 and R111K/STOP 155 constructs to a pentamannosyl phosphate-agarose affinity column was observed. 2 In contrast to that observed at positions 66, 111, 133, and 143, replacement of Tyr-45, His-105, or Arg-135 only partially inhibited binding of the CD-MPR to the pentamannosyl phosphate affinity columns, whereas substitutions at Asp-103 had no significant effect on Man-6-P binding (Table I and Fig. 3).…”

“…The mannose 6-phosphate receptors (MPRs) 1 function in the specific recognition of phosphorylated mannose residues, which have been identified on numerous proteins, including lysosomal enzymes (1,2), several growth factors (3,4), and a cytokine (5). Although an increasing number of different classes of proteins have been shown to interact with the receptors, the best characterized function of the MPRs in higher eukaryotic cells is their ability to target newly synthesized soluble acid hydrolases to the lysosome (6 -8).…”

An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities

“…A phosphorylated carbohydrate structure, most likely a mannose 6-phosphate residue on the lysosomal enzymes, is recognized by cell-surface receptors (Kaplan et al, 1977a,b;Ullrich et al, 1978). The effect of antibody binding on lysosomalenzyme recognition and endocytosis was assayed for a-N-acetylglucosaminidase prepared from human urine and 8-N-acetylglucosaminidase prepared from secretions of human skin fibroblasts.…”

“…Endocytosis of antibody-lysosomal-enzyme complexes a-N-Acetylglucosaminidase, partially purified from human urine (Ullrich et al, 1978), and fl-N-acetylglucosaminidase, prepared from fibroblast secretions (Hickman et al, 1974), were incubated with either the appropriate antiserum or control rabbit serum for 2h at 37°C and then for 16h at 4°C. After centrifugation, the supernatant of the controls and the precipitates of the antisera assays resuspended in 0.15M-NaCl were assayed for enzyme activity.…”

“…Equal activities from the resuspended precipitates and the supernatant of the controls were added to the culture medium of either mucopolysaccharidosis IIIB fibroblasts (deficient in a-N-acetylglucosaminidase) or Sandhoff fibroblasts (deficient in fl-Nacetylglucosaminidase). Enzyme endocytosis was determined as described by Ullrich et al (1978 (Kaltwasser et al, 1965) with bovine serum albumin as standard. Cell viability was monitored for each experimental condition by the Nigrosine test (Kaltenbach et al, 1958).…”