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

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 (αHS) 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. [35S]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. 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 (αHS) 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. [35S]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. IntroductionEndocytosis 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 macromolecular 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) 3The abbreviations used are: HSheparan sulfateHSPGheparan sulfate proteoglycanPGproteoglycanαHSscFv anti-HS antibodyHIVhuman immunodeficiency virusscFvsingle chain variable fragmentαHS-Ffluorescent αHS complexBSAbovine serum albuminCHOChinese hamster ovaryGPCglypicanGPIglycosyl-phosphatidyl-inositolNSN-sulfationPNSpostnuclear supernatantSDCsyndecan2-OS2-O sulfationGFPgreen fluorescent proteinDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineHAhemagglutinin. that are expressed on virtually all mammalian cells (1.Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 2.Belting M. Persson S. Fransson L.A. Biochem. J. 1999; 338: 317-323Crossref PubMed Scopus (90) Google Scholar, 3.Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Sandgren S. Wittrup A. Cheng F. Jönsson M. Eklund E. Busch S. Belting M. J. Biol. Chem. 2004; 279: 17951-17956Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5.Payne C.K. Jones S.A. Chen C. Zhuang X. Traffic. 2007; 8: 389-401Crossref PubMed Scopus (190) Google Scholar, 6.Wittrup A. Sandgren S. Lilja J. Bratt C. Gustavsson N. Mörgelin M. Belting M. J. Biol. Chem. 2007; 282: 27897-27904Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Together with the fact that several viruses utilize HSPGs for cell-surface adsorption and internalization (7.Spillmann D. Biochimie. 2001; 83: 811-817Crossref PubMed Scopus (147) Google Scholar, 8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), 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.Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar, 10.Bishop J.R. Schuksz M. Esko J.D. Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1239) Google Scholar) 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.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 9.Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar, 11.Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar, 12.Powell A.K. Yates E.A. Fernig D.G. Turnbull J.E. Glycobiology. 2004; 14: 17R-30RCrossref PubMed Scopus (223) Google Scholar). There are two major classes of HS-bearing cell-surface PGs: six members of the glypican (GPC) family of glycosyl-phosphatidyl-inositol (GPI)-linked proteins (13.Fransson L.A. Belting M. Cheng F. Jönsson M. Mani K. Sandgren S. Cell Mol. Life Sci. 2004; 61: 1016-1024Crossref PubMed Scopus (99) Google Scholar, 14.Filmus J. Capurro M. Rast J. Genome Biol. 2008; 9: 224Crossref PubMed Scopus (418) Google Scholar) and four members of the syndecan (SDC) family of transmembrane proteins (15.Couchman J.R. Nat. Rev. Mol. Cell Biol. 2003; 4: 926-937Crossref PubMed Scopus (336) Google Scholar, 16.Beauvais D.M. Rapraeger A.C. Reprod. Biol. Endocrinol. 2004; 2: 3Crossref PubMed Scopus (214) Google Scholar). 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 macromolecules (8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), 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 sequence, length, and net positive charge on the intracellular fate of delivered macromolecules, has been extensively studied (17.Nakase I. Niwa M. Takeuchi T. Sonomura K. Kawabata N. Koike Y. Takehashi M. Tanaka S. Ueda K. Simpson J.C. Jones A.T. Sugiura Y. Futaki S. Mol. Ther. 2004; 10: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 18.Nakase I. Tadokoro A. Kawabata N. Takeuchi T. Katoh H. Hiramoto K. Negishi M. Nomizu M. Sugiura Y. Futaki S. Biochemistry. 2007; 46: 492-501Crossref PubMed Scopus (337) Google Scholar). Indeed, the role of HSPGs in the uptake of macromolecular cargoes remains controversial. They have been proposed to act either as true internalizing receptors or as receptors for initial cell-surface attachment. In the latter case, ligand binding and concentration by the HSPG would result in subsequent ligand presentation to alternative, internalizing receptors or in ligand uptake through receptor-independent pinocytosis or even in direct membrane penetration through phospholipid interactions (5.Payne C.K. Jones S.A. Chen C. Zhuang X. Traffic. 2007; 8: 389-401Crossref PubMed Scopus (190) Google Scholar, 8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 18.Nakase I. Tadokoro A. Kawabata N. Takeuchi T. Katoh H. Hiramoto K. Negishi M. Nomizu M. Sugiura Y. Futaki S. Biochemistry. 2007; 46: 492-501Crossref PubMed Scopus (337) Google Scholar, 19.Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar).van Kuppevelt and co-workers (20.Dennissen M.A. Jenniskens G.J. Pieffers M. Versteeg E.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. J. Biol. Chem. 2002; 277: 10982-10986Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have previously described the development and characterization of epitope-specific, phage display-derived, scFv anti-HS antibodies (αHS) that can be utilized to probe the structural diversity of HS chains in various tissues and cell types. Here, using several of these αHS in conjunction with the development of a strategy for the isolation of endocytic vesicles, we address the internalizing function of cell-surface HSPG and investigate the respective roles of HS ligand epitope specificity and PG core protein specificity in macromolecular delivery.EXPERIMENTAL PROCEDURESMaterialsNa235SO4 (1310 Ci/mmol) was from PerkinElmer Life Sciences, HIV-Tat peptide (GRKKRRQRRRPPQC) was from Innovagen AB, Lund, Sweden, and YOYO-1 and fluorophore-labeled 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.Gonzalez A.D. Kaya M. Shi W. Song H. Testa J.R. Penn L.Z. Filmus J. J. Cell Biol. 1998; 141: 1407-1414Crossref PubMed Scopus (171) Google Scholar) constructs were kindly provided by Dr J. Filmus, University of Toronto, Canada. Full-length rat SDC2 and SDC3 cloned into pEGFP-N3 vector with a C-terminal GFP tag and intact sorting and glycosylation (22.Landgraf P. Wahle P. Pape H.C. Gundelfinger E.D. Kreutz M.R. J. Biol. Chem. 2008; 283: 25036-25045Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) 4P. Landgraf, personal communication. was a kind gift from Dr. P. Landgraf, Leibniz-Institute for Neurobiology, Magdeburg, Germany. αHS 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.ten Dam G.B. Hafmans T. Veerkamp J.H. van Kuppevelt T.H. J. Histochem. Cytochem. 2003; 51: 727-739Crossref PubMed Scopus (32) Google Scholar, 24.Smits N.C. Robbesom A.A. Versteeg E.M. van de Westerlo E.M. Dekhuijzen P.N. van Kuppevelt T.H. J. Cell Mol. Biol. 2004; PubMed Scopus Google Scholar). and mouse mouse and cell and and were from hamster cell X. Esko J.D. Mol. Biol. 2001; Google Scholar), human and lung cells were from the K.J. Esko J.D. Biochem. J. PubMed Scopus Google Scholar) and X. Esko J.D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) cells were kindly provided by Dr J. D. University of and cells were in and cells were in in a at using the respective with bovine and antibody cells were with with and with αHS and in and with mouse antibody that the epitope of αHS antibodies by in and with goat antibody antibody were in for on cells were in and by on a with uptake αHS mouse and goat antibodies were in at for and with cells at for were in bovine in and by αHS antibody were in all binding and uptake HS were either with AO4B08 and mouse antibody or with AO4B08 and antibody for various of at cells were with the ligands for at were with to ligands, and with by with AO4B08 or as described antibody cells were with or both GFP and using to the and for to for uptake for was determined in and cells by cells were with with to the cells in were with at for various of were with to ligand and in was with In of cells with or both and HA-GPC3 were with AO4B08 and antibody for at with and for in and with goat and or with mouse by goat and goat antibodies or with mouse by goat and goat antibodies and cells were with and mouse with and antibody for at and with goat and goat were in and using with a mouse and magnetic MagCellect goat anti-mouse antibodies were to in at for was to cells at for various of by with in bovine and with was in all subsequent were by an including through a during the of and and for at intact cell and were by at for was with the obtained a at for to a was into a magnetic and in a magnetic were using an To the and magnetic of the from cells with and were using a with a or using a cell with the to of cells with at for various of were with by in bovine in and for at and at in in were with for at in in in a of and in using as an were into on an and with and isolated membrane vesicles were and as or by negative and as described previously J. H. PubMed Scopus (136) Google Scholar). were in a at were with a and of cells were with in for Cell were extensively with and with at for various of and the of cells was into and magnetic as described was concentration and were for at were purified using and by and or by in a as described previously M. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In some isolated were with as described M. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), to were using and by using proteins were to membrane with mouse or provided by Dr. K. goat or mouse a kind gift from Dr. A. antibody by the antibody were using and cellular was using mammalian and was using was used for on in an of PG core protein expression as with was on of and are of at independent in in and are the In some and the were the and HIV-Tat compete for HS and both internalization of cell-surface cell-surface binding of in cells in the of of AO4B08 antibody uptake of a of in the of of AO4B08 antibody and co-localize in endocytic vesicles. analysis of cells for with and and is cells were with various internalizing HS ligands, i.e. AO4B08 antibody peptide or for the at with to the ligands, the of cell-surface HSPG was determined by with AO4B08 antibody at by cells were with or AO4B08 antibody (αHS) for at with the of cell-surface AO4B08 and epitope was determined by with antibodies at by cells were for at with AO4B08 antibody and antibody and using of the in to the has been that cell-surface HSPGs are M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the role of HSPG during macromolecular delivery remains Here, we provide the first biochemical evidence that HSPGs are true internalizing receptors of macromolecular and for the first that both SDC and GPC HSPG can this we that is epitope-specific, i.e. only among several cell-surface-binding αHS was efficiently should be that as to ligands, e.g. and that be internalized through HSPG high binding the on HSPG for αHS internalization to probe the HSPG endocytic AO4B08 antibody has shown for HS S. Jenniskens G.J. K. H. Li J.P. Lindahl U. van Kuppevelt T.H. Spillmann D. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google cell virtually uptake of AO4B08 structural specificity of AO4B08 uptake is as has been shown that the in HS chains with iduronic acid and a sulfation as with to X. Esko J.D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of the αHS antibodies to HSPG was induction of HSPG internalization was shown to the HS epitope of a antibody from the cell and AO4B08 and antibody were shown to in is that the of AO4B08 to macromolecular is to HS epitope of a specific of PG core Instead, the of HS e.g. to or from the core protein provide a to HSPG and membrane in cells of HSPGs X. K.J. H. K. Esko J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), that of AO4B08 uptake by this cell is to HSPG of the AO4B08 antibody to magnetic uptake through HSPG was utilized for the isolation of intact this we that both SDC and GPC are with macromolecular data indicate at an of ligand GPC and SDC can in a SDC and GPC can be αHS their structural M. M. J. M. Rev. Biochem. 1999; PubMed Scopus Google Scholar), has been by previous studies have that and core protein antibodies SDC into E. E. R.V. M. J. Cell Sci. 2004; PubMed Scopus Google Scholar). SDC in with GPC in microdomains for subsequent of their intracellular have a more direct role in signaling e.g. through interactions with to membrane and macromolecular is macromolecular of GPC endocytic interactions with membrane cell-surface signaling data from that AO4B08 antibody cells through endocytosis. P. M. and M. in studies should provide a detailed characterization of isolated endocytic vesicles of this that internalized HSPG remains intact for more in isolated vesicles is that are to be for and that with specific HS and is M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Our data that core protein internalized HSPGs to an sorting that antibody the of by latter is in with the that iduronic acid are important for of HS X. K.J. H. K. Esko J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). antibody with the of HS chains at several and at a detailed structural characterization of that several αHS to cell-surface HSPG were internalized and that AO4B08 was by cell from various the of a or HS for drug delivery for the in this have several delivery of a or delivery for that target a that is for cell for of the HSPG in macromolecular important for the development of drug delivery IntroductionEndocytosis 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 macromolecular 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) 3The abbreviations used are: HSheparan sulfateHSPGheparan sulfate proteoglycanPGproteoglycanαHSscFv anti-HS antibodyHIVhuman immunodeficiency virusscFvsingle chain variable fragmentαHS-Ffluorescent αHS complexBSAbovine serum albuminCHOChinese hamster ovaryGPCglypicanGPIglycosyl-phosphatidyl-inositolNSN-sulfationPNSpostnuclear supernatantSDCsyndecan2-OS2-O sulfationGFPgreen fluorescent proteinDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineHAhemagglutinin. that are expressed on virtually all mammalian cells (1.Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 2.Belting M. Persson S. Fransson L.A. Biochem. J. 1999; 338: 317-323Crossref PubMed Scopus (90) Google Scholar, 3.Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Sandgren S. Wittrup A. Cheng F. Jönsson M. Eklund E. Busch S. Belting M. J. Biol. Chem. 2004; 279: 17951-17956Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5.Payne C.K. Jones S.A. Chen C. Zhuang X. Traffic. 2007; 8: 389-401Crossref PubMed Scopus (190) Google Scholar, 6.Wittrup A. Sandgren S. Lilja J. Bratt C. Gustavsson N. Mörgelin M. Belting M. J. Biol. Chem. 2007; 282: 27897-27904Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Together with the fact that several viruses utilize HSPGs for cell-surface adsorption and internalization (7.Spillmann D. Biochimie. 2001; 83: 811-817Crossref PubMed Scopus (147) Google Scholar, 8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), 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.Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar, 10.Bishop J.R. Schuksz M. Esko J.D. Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1239) Google Scholar) 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.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 9.Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar, 11.Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar, 12.Powell A.K. Yates E.A. Fernig D.G. Turnbull J.E. Glycobiology. 2004; 14: 17R-30RCrossref PubMed Scopus (223) Google Scholar). There are two major classes of HS-bearing cell-surface PGs: six members of the glypican (GPC) family of glycosyl-phosphatidyl-inositol (GPI)-linked proteins (13.Fransson L.A. Belting M. Cheng F. Jönsson M. Mani K. Sandgren S. Cell Mol. Life Sci. 2004; 61: 1016-1024Crossref PubMed Scopus (99) Google Scholar, 14.Filmus J. Capurro M. Rast J. Genome Biol. 2008; 9: 224Crossref PubMed Scopus (418) Google Scholar) and four members of the syndecan (SDC) family of transmembrane proteins (15.Couchman J.R. Nat. Rev. Mol. Cell Biol. 2003; 4: 926-937Crossref PubMed Scopus (336) Google Scholar, 16.Beauvais D.M. Rapraeger A.C. Reprod. Biol. Endocrinol. 2004; 2: 3Crossref PubMed Scopus (214) Google Scholar). 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 macromolecules (8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), 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 sequence, length, and net positive charge on the intracellular fate of delivered macromolecules, has been extensively studied (17.Nakase I. Niwa M. Takeuchi T. Sonomura K. Kawabata N. Koike Y. Takehashi M. Tanaka S. Ueda K. Simpson J.C. Jones A.T. Sugiura Y. Futaki S. Mol. Ther. 2004; 10: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 18.Nakase I. Tadokoro A. Kawabata N. Takeuchi T. Katoh H. Hiramoto K. Negishi M. Nomizu M. Sugiura Y. Futaki S. Biochemistry. 2007; 46: 492-501Crossref PubMed Scopus (337) Google Scholar). Indeed, the role of HSPGs in the uptake of macromolecular cargoes remains controversial. They have been proposed to act either as true internalizing receptors or as receptors for initial cell-surface attachment. In the latter case, ligand binding and concentration by the HSPG would result in subsequent ligand presentation to alternative, internalizing receptors or in ligand uptake through receptor-independent pinocytosis or even in direct membrane penetration through phospholipid interactions (5.Payne C.K. Jones S.A. Chen C. Zhuang X. Traffic. 2007; 8: 389-401Crossref PubMed Scopus (190) Google Scholar, 8.Belting M. Trends Biochem. Sci. 2003; 28: 145-151Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 18.Nakase I. Tadokoro A. Kawabata N. Takeuchi T. Katoh H. Hiramoto K. Negishi M. Nomizu M. Sugiura Y. Futaki S. Biochemistry. 2007; 46: 492-501Crossref PubMed Scopus (337) Google Scholar, 19.Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar).van Kuppevelt and co-workers (20.Dennissen M.A. Jenniskens G.J. Pieffers M. Versteeg E.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. J. Biol. Chem. 2002; 277: 10982-10986Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have previously described the development and characterization of epitope-specific, phage display-derived, scFv anti-HS antibodies (αHS) that can be utilized to probe the structural diversity of HS chains in various tissues and cell types. Here, using several of these αHS in conjunction with the development of a strategy for the isolation of endocytic vesicles, we address the internalizing function of cell-surface HSPG and investigate the respective roles of HS ligand epitope specificity and PG core protein specificity in macromolecular delivery.