Abstract
Chinese hamster ovary cells were transfected with a recombinant DNA containing the entire coding sequence of human lysosomal protective protein cDNA under the control of mouse metallothionein I promoter. Neomycin and methotrexate-resistant stably transformed cell lines expressing this protein were isolated. Immunoprecipitation of the product with antiserum against human placental protective protein-beta-galactosidase complex revealed a 52-kDa protective protein precursor, which was then processed to mature form, a heterodimer of 32- and 20-kDa polypeptides. The precursor secreted in the culture medium was taken up by the mannose 6-phosphate receptor system and restored acid carboxypeptidase, beta-galactosidase, and neuraminidase activities in galactosialidosis fibroblasts. The expressed protein showed a granular pattern in intracellular distribution, was fractionated at the density of lysosomes, and had serine esterase activities; acid carboxypeptidase at pH 5.6, esterase at pH 7.0, and carboxyl-terminal deamidase at pH 7.0. They were inhibited simultaneously by phenylmethylsulfonyl fluoride, N-benzyloxycarbonyl-L-phenylalanine chloromethyl ketone, or iodoacetamide. The acid carboxypeptidase activity of the purified monomeric mature protective protein was labile in vitro under the acidic condition. Saposins (sphingolipid activator proteins) stabilized the activity at micromolar level concentrations.
Highlights
RecombinantDNA containing the entire coding se- This glycoprotein is processed in human fibroblasts from a quence of human lysosomal protective protein cDNA precursor to a matureform, a heterodimer of two polypeptides under the control of mousemetallothioneinI promoter. held together by disulfide bonds [2,4], andaggregateswith 8
The precursorsecreted in the culture medium was takenup by the mannose 6-phosphate receptor system and restored acid carboxypeptidase, &galactosidase, and involving neuraminidase [5,6,7]
The precursor protein secreted from normal fibroblasts treated with ammonium chloride is taken up by galactosialidosis fibroblasts via the mannose 6-phosphate receptor system and restores p-galactosidase and neuraminidase activities [1, 2]
Summary
Cells-The procedures were performed as described by Kyle et al. (19) with minor modifications. Preparation of Protective Protein Precursor-The stably transformed CHO cells were seeded on 125-mm dishes in subconfluency and cultured for 24 h. They were washed with PBS twice and further cultured in 25 ml of FCS-free Ham's F-10 containing 10 mM ammonium chloride. Purification of Mature Protective Protein-One-ninth volume of 0.25 M imidazole HC1 buffer (pH 7.4) was added to the cell extract prepared as described above, and the suspension (12 ml) was loaded (120 ml/h) on a chromatofocusing column (Pharmacia PBE94, 0.9 X 30 cm) preequilibrated with 25 mM imidazole HCl buffer (pH 7.4) at 4 "C. Immunoprecipitation of Protective Protein-Subconfluent trans- times by concentration and dilution with 30 volumesof 20 mM sodium formed CHO cells were incubated for 1h inmethionine/cysteine-free phosphate buffer (pH 7.2) in Centriprep 30 (Amicon).
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