Introduction of purified hexosaminidase A into Tay-Sachs leukocytes by means of immunoglobulin-coated liposomes.
To determine whether ligand-receptor interactions could engender the selective uptake by deficient cells of enzyme-laden liposomes, aggregated human IgG was used to coat liposomes which had previously trapped purified hexosaminidase A (Hex A). By a new, high-yield procedure, Hex A was purified 7000-fold from human placenta: the homogeneous protein had a pI of 5.4, permitting nonelectrostatic trapping in the aqueous interstices of anionic multilamellar liposomes (molar ratios of phosphatidyl-choline-dicetyl phosphate-cholesterol, 7:2:1). Trapped Hex A was separated from free enzyme by means of Sephadex G-200 chromatography: 1.3 +/- 0.3 mUnits of Hex A/mumol of phospholipid became associated with liposomes and trapped glucose, utilized as a marker of the aqueous compartment. Once sequestered, the enzyme remained latent until lamellae were disrupted by Triton X-100. Presence of enzyme in aqueous compartments was proved by the demonstration of increased trapping (0.02-1.33 mUnits/mumol of phospholipid) with increments in like-sign repulsion of the bilayers produced by increasing molar ratios of anionic dicetyl phosphate (5-20%). To provide for ligand-receptor interaction with surface Fc receptors of human polymorphonuclear leukocytes (PMN's), liposomes were coated by heat-aggregated (62 degrees C, 10 min) human IgG. PMN's from Tay-Sachs patients genetically deficient in Hex A activity readily incorporated exogenous Hex A provided in this fashion. PMN's exposed to enzyme-laden liposomes coated with aggregated IgG incorporated significantly more Hex A than when the enzyme was presented in uncoated liposomes or in liposomes coated with native IgG, which engages Fc receptors with less avidity. Free enzyme was not endocytized. Acquisition of specific Hex A isozyme activity by cells (determined by DEAE-cellulose chromatography) was not due to surface adsorption since cytochalasin B, which prevents phagocytosis but not surface adherence; blocked uptake. Incorporation of the isozyme by deficient cells was also demonstrated by starch gel electrophoresis, and ultrastructural studies showed that the immunoglobulin-coated, Hex A-containing liposomes were taken up into PMN lysosomes after membrane fusion. The studies indicate that liposomes coated with surface ligands may be used to introduce enzyme or other materials into deficient cells possessing appropriate surface receptors.
D001692:Biological Transport; D002784:Cholesterol; D005260:Female; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism* / Q000648:ultrastructure; D008064:Lipidoses / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008854:Microscopy, Electron; D010713:Phosphatidylcholines; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011955:Receptors, Drug
D007074:Immunoglobulin G; D008081:Liposomes; D010713:Phosphatidylcholines; D011955:Receptors, Drug; D002784:Cholesterol; D006596:Hexosaminidases