Abstract
Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular 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 immunodeficiency 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 superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-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.
Highlights
Endocytosis regulates many processes, including signaling events involved in cell motility and cell fate determination, nutrient uptake, microbial invasion, and macromolecular drug delivery
To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [35S]sulfate-labeled heparan sulfate proteoglycans (HSPGs) was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand
Previous reports have documented an important role of HSPG in cellular internalization of numerous macromolecules [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
Summary
Na235SO4 (1310 Ci/mmol) was from PerkinElmer Life Sciences, HIV-Tat peptide (GRKKRRQRRRPPQC) was from Innovagen AB, Lund, Sweden, and YOYO-1 and fluorophorelabeled antibodies were from Invitrogen. MagCellect magnetic nanoparticle-conjugated goat anti-mouse IgG was from R&D Systems. N-terminally tagged full-length rat HA-GPC3 [21] constructs were kindly provided by Dr J. Full-length rat SDC2 and SDC3 cloned into pEGFP-N3 vector with a C-terminal GFP tag and intact sorting and glycosylation [22]4 was a kind gift from Dr P. ␣HS antibodies were obtained by biopanning against HS isolated from bovine kidney (HS4E4, HS4C3), from skeletal muscle from mouse (AO4B08) and human (RB4EA12), and from human lung (EV3C3) as described [23, 24]. Chondroitinase ABC lyase, heparanases I and III, mouse monoclonal anti-vsv (P5D4), mouse monoclonal anti-myc (9E10), and rabbit polyclonal anti-vsv antibodies, cell media and supplements, and fine grade chemicals were from Sigma
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