ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles

Abstract

Cellular uptake of several viruses and polybasic macromole-cules requires the expression of cell-surface heparan sulfate pro-teoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (HS) clones, only one, AO4B08, efficiently translocated macromolec-ular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human im-munodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamag-netic nanoparticles. [ 35 S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internaliza-tion with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phos-phatidyl-inositol-anchored glypican in purified vesicles. Importantly , internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein. Endocytosis regulates many processes, including signaling events involved in cell motility and cell fate determination, nutrient uptake, microbial invasion, and macromolecular drug delivery. Cellular internalization of highly diverse macro-molecular ligands, including polylysine/cationic lipid-DNA complexes, cationic polymers, antimicrobial peptide-DNA complexes, and "naked" DNA, have been shown to depend on cell-surface heparan sulfate proteoglycans (HSPGs) 3 that are expressed on virtually all mammalian cells (1-6). Together with the fact that several viruses utilize HSPGs for cell-surface adsorption and internalization (7, 8), HSPG emerges as an important target molecule that should be considered in the development of drug delivery vehicles. HSPGs are a class of proteins substituted with glucosamine-glucuronic acid disaccharide polysaccharides (9, 10) that are extensively modified during/subsequent to polymerization, including N-deacetylation/sulfation and 6-and 3-O-sulfation of the glucosamine, epimerization at C-5 of glucuronic acid into iduronic acid, and 2-O-sulfation (2-OS) of iduronic acid. These modifications provide the HS chains with complex patterns of sulfation and high negative charge that largely determine their functional interactions with polybasic ligands (8, 9, 11, 12). There are two major classes of HS-bearing cell-surface PGs: six members of the glypican (GPC) family of glycosyl-phosphati-dyl-inositol (GPI)-linked proteins (13, 14) and four members of the syndecan (SDC) family of transmembrane proteins (15, 16). Due to their differential modes of membrane association, GPC and SDC are generally believed to present HS chains to different plasma membrane microdomains to exert specific biological functions. Although previous reports have documented an important role of HSPG in cellular internalization of numerous macro-molecules (8), some key questions on the exact function of HSPG in this process have remained unanswered, i.e. few studies have focused on the fate of cell-surface HSPG and the specific roles of SDC and GPC during macromolecular delivery. Instead, the influence of HS ligand properties, e.g. peptide