P-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 2. Biological properties.

Abstract

A conjugate of p-aminophenyl 6-phospho-alpha-D-mannopyranoside and bovine serum albumin was shown to interact with the uptake system for lysosomal enzymes in cultured human diploid fibroblasts. Radioiodinated conjugate containing 20 mol of mannose 6-phosphate/mol of albumin was taken up by the cells and degraded to trichloroacetic acid soluble fragments which were released into the medium. Unlabeled conjugate, mannose 6-phosphate, and a lysosomal enzyme, L-iduronidase, inhibited the uptake of the 125I-labeled conjugate (Ki = 2 X 10(-8), 5 X 10(-6), and 1.5 X 10(-9) M, respectively). Conversely, the uptake of L-iduronidase was competitively inhibited by the mannose 6-phosphate conjugate as well as by free mannose 6-phosphate; however, higher concentrations of these compounds were required (Ki = 10(-6) and 5 X 10(-5) M, respectively). These results suggest that although L-iduronidase and the conjugate are bound to the same receptor by mannose 6-phosphate residues, the uptake of the enzyme involves some additional structure that is not shared by the conjugate. Internalization of the radiolabeled mannose 6-phosphate albumin conjugate was observed only in human diploid fibroblast strains. An SV-40 transformed line of human fibroblasts as well as three permanent rodent fibroblast lines (CHO, NRK, and L cells) failed to take up the conjugate, presumably because they were deficient in receptors or in the ability to internalize receptor-conjugate complexes.