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• W2053873441 abstract "Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA. Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA. IntroductionProteoglycans are biologically important molecules that are ubiquitously expressed on the cell surfaces as well as the extracellular and intracellular spaces and are involved in a variety of functions including tissue organization, morphogenesis, cellular communication, adhesion, migration, growth, and signaling to name few (1Bishop J.R. Schuksz M. Esko J.D. Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1239) Google Scholar). They are made up of a protein core linked to linear polysaccharide chains called glycosaminoglycans (GAGs). 3The abbreviations used are: GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; C6S, chondroitin 6-sulfate. These glycosaminoglycan chains are composed of different repeating disaccharide units that are sulfated at various positions. Based on the nature of the repeating units and the degree of sulfation, heparin, heparan sulfate, chondroitin sulfates, keratan sulfates, and hyaluronan are the different types of GAGs well known in the literature. Sulfated proteoglycans on the cell membranes are the major contributors to the negative charge on the cell surface, making it available for electrostatic interactions. These proteoglycans bind many ligands like growth factors and chemokines as well as large proteins. They have also been implicated in the internalization of foreign molecules and organisms through endocytotic pathways. Many viruses including adeno-associated virus (2Summerford C. Samulski R.J. J. Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar), herpes simplex virus, human papilloma virus, and human immunodeficiency virus (3Chen Y. Götte M. Liu J. Park P.W. Mol. Cells. 2008; 26: 415-426PubMed Google Scholar) initially bind to cell surface heparan sulfate proteoglycans to mediate their cellular entry.Non-viral gene delivery vectors like lipoplexes (lipid-DNA complexes) and polyplexes (polymer-DNA complexes), which are usually cationic in nature, can also interact with both cell surface and extracellular GAGs through electrostatic interactions. The cell surface GAGs have been thought to act as the primary receptor for the attachment of the cationic complexes followed by endocytosis of the complexes (for review, see Ref. 4Poon G.M. Gariépy J. Biochem. Soc. Trans. 2007; 35: 788-793Crossref PubMed Scopus (6) Google Scholar). Early reports showed that poly-l-lysine-DNA complexes as well as different cationic liposome-DNA complexes require cell surface proteoglycans (specifically, heparan sulfate proteoglycans) for delivery of DNA both in vitro and in vivo (5Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 6Mounkes L.C. Zhong W. Cipres-Palacin G. Heath T.D. Debs R.J. J. Biol. Chem. 1998; 273: 26164-26170Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). However, many subsequent reports have shown that their function in cellular entry of non-viral vector complexes may be dispensable, and strong binding of cationic complexes to either cell surface or extracellular GAGs may actually prevent their uptake. It was demonstrated that cellular uptake of cationic lipoplexes occurred at similar levels in cell lines with cell surface GAGs as well as in GAG-deficient cell lines (7Belting M. Petersson P. Biochem. J. 1999; 342: 281-286Crossref PubMed Scopus (39) Google Scholar), which implies that any role of GAGs in controlling transfection efficiency can at best come into play at a later step of the transfection process. It has also been suggested that cell surface proteoglycans protect cells from the cytotoxic effects of cationic lipids, and hence, proteoglycan-deficient cell lines give lowered transfection at high lipid to DNA ratios (7Belting M. Petersson P. Biochem. J. 1999; 342: 281-286Crossref PubMed Scopus (39) Google Scholar). In contrast, another study showed that cationic polymers such as poly-l-lysine and polyethyleneimine, and lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) and 1,2-dioleyl-3-phosphatidylethanolamine (DOPE) show strong binding to the cell surface GAGs but decreased transfection efficiency in their presence due to decreased cellular uptake (8Ruponen M. Honkakoski P. Tammi M. Urtti A. J. Gene Med. 2004; 6: 405-414Crossref PubMed Scopus (91) Google Scholar). The exact role of the cell surface proteoglycans in lipoplex or polyplex delivery is, thus, elusive, and different experimental conditions as well as different chemical and structural characteristics of the cationic carriers might be responsible for the differences observed in the data.Exogenous addition of negatively charged GAGs in vitro during transfection has also been studied in most of these systems and has usually been found to decrease the gene transfer efficiency (5Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 9Ruponen M. Rönkkö S. Honkakoski P. Pelkonen J. Tammi M. Urtti A. J. Biol. Chem. 2001; 276: 33875-33880Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). It is thought that depending on the chemistry of the carrier, the nature of the complex, and the charge density of GAGs, the decrease in transfection may be due to one or all of the following reasons. (i) Sulfated GAGs cause premature extracellular release of DNA from its complexes with cationic polymers (10Ruponen M. Ylä-Herttuala S. Urtti A. Biochim. Biophys. Acta. 1999; 1415: 331-341Crossref PubMed Scopus (305) Google Scholar, 11Burke R.S. Pun S. Bioconjugate Chem. 2008; 19: 693-704Crossref PubMed Scopus (170) Google Scholar) or cationic lipids (12Zelphati O. Szoka Jr., F.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11493-11498Crossref PubMed Scopus (589) Google Scholar). (ii) Soluble GAGs could compete with cell surface proteoglycans for binding to the complex, thereby decreasing cellular uptake (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). (iii) Binding of GAGs may change the endocytotic uptake route of the complex. (iv) GAGs may cause altered intracellular distribution of the complexes, significantly affecting the gene expression (9Ruponen M. Rönkkö S. Honkakoski P. Pelkonen J. Tammi M. Urtti A. J. Biol. Chem. 2001; 276: 33875-33880Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).The strong and universal translocation of arginine-rich cell-penetrating peptides in multiple cell types has also triggered speculation on the involvement of some common molecules like cell surface GAGs in the process of cellular entry, although it is still debatable whether membrane translocation and endocytotic uptake are both involved (14Futaki S. Nakase I. Tadokoro A. Takeuchi T. Jones A.T. Biochem. Soc. Trans. 2007; 35: 784-787Crossref PubMed Scopus (187) Google Scholar). Cell-penetrating peptides like naturally occurring protein transduction domains, e.g. the HIV-TAT peptide (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar), Antennapedia peptide (15Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), penetratin, and even synthetic homoarginines of certain compositions (16Fuchs S.M. Raines R.T. Biochemistry. 2004; 43: 2438-2444Crossref PubMed Scopus (320) Google Scholar, 17Nakase 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 (336) Google Scholar), are likely to involve cell surface GAGs in their cellular entry. It has also been seen that when these peptides are used for cargo delivery, the requirement of cell surface GAGs differs. Cellular uptake of HIV-1 TAT peptide conjugated to cargo was first surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans, which was confirmed by impaired uptake on enzymatic or genetic removal of the cell surface GAGs (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). However, uptake of free TAT peptide was suggested to involve either different receptors or pathways because its internalization was not completely inhibited in cells lacking surface heparan sulfate (19Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). The involvement of cell surface proteoglycans on the cellular entry of the peptide and its complexes with cargo can be affected by many factors such as net charge of the complex (18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) as well as the structure and distribution of positive charges on the peptides (17Nakase 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 (336) Google Scholar), and hence, it is possible that the bare peptide and the complex behave in different ways. More recently, it was proposed that transduction mediated by TAT can occur in glycan-deficient cell lines and the cell surface glycans bind TAT and only affect the efficiency of transduction (20Gump J.M. June R.K. Dowdy S.F. J. Biol. Chem. 2010; 285: 1500-1507Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, although arginine-rich peptides have high affinity to sulfated GAGs, especially heparan sulfate, and heparin (21Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and could co-internalize heparan sulfate, mediating nuclear delivery (18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), soluble sulfated GAGs inhibited cargo delivery by arginine-rich peptides (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 15Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). All these evidences suggest that the role of GAGs in controlling cellular entry of cationic peptides with and without cargo needs further elucidation.Under this backdrop, we have explored the role of both cell surface and soluble GAGs during gene delivery using model peptide carriers, arginine, and lysine homopeptides (each 16-mer in length). Peptides containing these two amino acids either alone or in different proportions (e.g. TAT peptide) and often with simple chemical modifications like attachment of other additional amino-acids, polyethylene glycol, functional moieties like targeting ligands, and fusogenic peptides, etc. are the most commonly studied peptide based gene delivery agents (22Martin M.E. Rice K.G. AAPS J. 2007; 9: E18-E29Crossref PubMed Scopus (255) Google Scholar, 23Mann A. Thakur G. Shukla V. Ganguli M. Drug Discov. Today. 2008; 13: 152-160Crossref PubMed Scopus (56) Google Scholar). We show that the 16-mer arginine and lysine homopeptides differ in their requirement of cell surface GAGs as well as in their interaction with soluble GAGs during gene delivery. This is reflected in the alterations of complex morphology, pattern of cellular binding, internalization, and eventual gene delivery efficiency between the two peptide systems. Our results highlight the importance of peptide chemistry as a key determinant for requirement of GAGs in gene delivery.DISCUSSIONCell surface glycosaminoglycans, by virtue of their negative charge density, are known to act as initial attachment sites or receptors for the internalization of a variety of ligands. However, although these molecules seem to be important in the entry of cell penetrating peptides like TAT, Antp, and some oligoarginines, a general consensus on their exact role is lacking. Multiple factors seem to affect the interaction between the positively charged peptides and the cell surface GAGs and eventually their internalization mechanism. The peptide sequence, charge distribution and the extracellular peptide concentration are the major influential factors (28Jiao C.Y. Delaroche D. Burlina F. Alves I.D. Chassaing G. Sagan S. J. Biol. Chem. 2009; 284: 33957-33965Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) along with the composition of the cell surface or the presence of other receptors. It has also been observed that the peptide conjugated to a cargo, either covalently or electrostatically, behave differently or may have alternative internalization mechanisms as compared with the free peptide (19Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). With respect to the peptide complexes with cargo molecules like DNA, the role of cell surface GAGs is ambiguous and seems to affect various stages of cellular entry like complex stability and cell surface binding as well as intracellular processing. Moreover, free exogenous GAGs may hinder the gene delivery efficiency by peptide-DNA complexes, as has already been reported for lipoplexes and polyplexes (5Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 9Ruponen M. Rönkkö S. Honkakoski P. Pelkonen J. Tammi M. Urtti A. J. Biol. Chem. 2001; 276: 33875-33880Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).In this study, our aim was to explore the role of cell surface and exogenous GAGs on the DNA delivery efficiency of arginine and lysine homopeptides of similar length, with the main focus on the role of peptide chemistry in this process. These two amino acids are the most common residues in peptide-based carriers for gene delivery and were thus obvious choices for a comparative study. Our results indicate that arginine- and lysine-based peptides differ in their requirement of cell surface GAGs for both cellular entry as well as DNA delivery. In the case of R16, the free peptide and the polyplex appear to adopt different routes of internalization. The arginine peptide enters entirely by non-endocytotic pathway as observed by near complete internalization at low temperature. On the other hand, R16 polyplexes go through an endocytotic mechanism. However, it is interesting to note that in both cases, there is very little difference in the uptake or the transfection efficiency between the wild type and the glycan-deficient cell lines or under the conditions of enzymatic removal of cell surface GAGs (as seen from FIGURE 1, FIGURE 2), indicating that the peptide and polyplex probably utilize different entry paths, which are both non-GAG-dependent. In contrast, both the bare lysine homopeptide and its complex are taken up into the cells primarily through an endocytotic route of entry involving cell surface GAGs. There is a reduction in the uptake of both the K16 peptide and polyplex in the mutant cell line and a decrease in the transfection efficiency on enzymatic removal of cell surface GAGs (both HS and CS). This implicates GAGs as primary attachment sites for the polyplexes, which is confirmed by poor cell association and internalization at low temperature in the mutant cell line. However, the transfection efficiency in the mutant cell line is not hugely altered. This apparent discrepancy could arise from a step beyond the entry process, i.e. possible routing through a different intracellular processing pathway in the mutant cell line as compared with the wild type, leading to efficient gene expression. Altogether, these results indicate that a glycan-dependent pathway of entry seems to be the predominant mode for the lysine peptide.The cellular entry of arginine-rich peptides has been proposed to occur by multiple pathways, including direct translocation across the membrane and endocytosis (29Duchardt F. Fotin-Mleczek M. Schwarz H. Fischer R. Brock R. Traffic. 2007; 8: 848-866Crossref PubMed Scopus (644) Google Scholar, 30Nakase I. Takeuchi T. Tanaka G. Futaki S. Adv. Drug Delivery Rev. 2008; 60: 598-607Crossref PubMed Scopus (300) Google Scholar). Both the pathways were operational in the proteoglycan-deficient cell line as well, although at higher peptide concentration, showing that the cell surface proteoglycans are dispensable for cellular entry of arginine homopeptides (31Kosuge M. Takeuchi T. Nakase I. Jones A.T. Futaki S. Bioconjugate Chem. 2008; 19: 656-664Crossref PubMed Scopus (262) Google Scholar). The polar, positively charged arginine oligomers can recruit negatively charged membrane components such as fatty acids to transiently produce a less polar ion pair complex that can partition into the lipid bilayer and thus translocate across the plasma membrane, driven by the membrane potential (32Rothbard J.B. Jessop T.C. Wender P.A. Adv. Drug Delivery Rev. 2005; 57: 495-504Crossref PubMed Scopus (234) Google Scholar). This is due to the more effective bidentate hydrogen bonding possible for guanidino groups in arginine versus the mondentate hydrogen bonding for ammonium groups in lysine. Therefore, lysine peptides form weaker interactions with membrane components, and direct translocation of lysine is not efficient (32Rothbard J.B. Jessop T.C. Wender P.A. Adv. Drug Delivery Rev. 2005; 57: 495-504Crossref PubMed Scopus (234) Google Scholar, 33Mitchell D.J. Kim D.T. Steinman L. Fathman C.G. Rothbard J.B. J. Pept. Res. 2000; 56: 318-325Crossref PubMed Scopus (890) Google Scholar). It is possible that such a mechanism of entry is operative here as well in case of the arginine peptides and polyplexes, whereas the lysine counterpart employs a GAG-dependent endocytotic route.We have also checked the transfection efficiency of the two peptides in the presence of exogenous soluble GAGs to explore whether there are differences in the interaction of arginine and lysine peptides with GAGs. Surprisingly, we observed that in the presence of exogenous HS and CS, the transfection efficiency of R16 increases by an order of magnitude or more under certain concentrations of GAGs (Fig. 3), which is contrary to some of the earlier reports. The increase is seen at relatively low GAG:peptide w/w ratios of 0.1:1 and 0.5:1 and is independent of cell surface GAGs. At relatively higher amounts of GAGs, there is a decrease in transfection, as has been observed earlier, possibly by destabilization of the complexes (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 15Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). On the other hand, in the case of K16 polyplexes, the addition of exogenous GAGs does not in general cause any major increase in the transfection efficiency and shows considerable drop at most of the GAG concentrations used.To explain this, we analyzed the stability of these polyplexes on anionic challenge. In vitro analysis of the stability of the polyplexes in the presence of soluble GAGs by agarose gel electrophoresis and ethidium bromide exclusion assay shows that both types of polyplexes are destabilized only at very high amounts of HS or CS (significantly higher than the conditions under which increased transfection is seen). The extent of destabilization is higher in the case of K16 polyplexes. However, atomic force microscopy reveals that there are actually subtle changes in the polyplex morphology in the two cases in the presence of GAGs. R16 complexes show altered morphology in the presence of GAGs but do not release DNA, whereas the K16 polyplexes clearly release DNA at all concentrations of anionic challenge despite the fact that arginines are known to bind more strongly to GAGs than lysines (34Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. Bioessays. 1998; 20: 156-167Crossref PubMed Scopus (514) Google Scholar). DNase I protection assay also confirms that low amounts of GAGs confer stability to the R16 polyplexes making the compacted DNA unavailable for nuclease degradation, as compared with the native polyplexes without GAGs. However, such protection is not offered in the case of K16 polyplexes. Thus, clearly the K16 complexes, by virtue of their loosened nature, are easily disassembled in presence of exogenous GAGs, which could be the major reason for their lowered transfection in such conditions.The differences in the interaction of arginine and lysine polyplexes with soluble GAGs can be ascribed to differences in the nature of the complexes formed by these peptides with DNA. We have earlier observed in a separate study that arginine- and lysine-based peptides have different DNA delivery efficiency due to differences in their DNA compaction and release mechanism. At a charge ratio 10, where maximum transfection efficiency (with least toxicity) is seen with both the peptides, the polyplexes show small size and uniform size distribution in the case of R16, whereas the particles are loosely packed in case of K16 (this is also shown in Fig. 5 under conditions where GAGs are absent). We have attributed this to the fact that K16 shows multiple modes of complexation (both monomolecular and multimolecular pathways are operative) as compared with R16 (where only multimolecular pathways are seen). The release of DNA from the polyplexes on anionic challenge also corroborates this, as K16 is more easily destabilized in the presence of anionic agents.4 It has also been shown in the literature that arginine polypeptide binds with higher affinity to different glycosaminoglycans like heparan sulfate and chondroitin sulfate than lysine, as the guanidino group of arginine can form electrostatic interaction as well as hydrogen bonds with the sulfate groups in GAGs when compared with the ammonium cation of lysine (25Verrecchio A. Germann M.W. Schick B.P. Kung B. Twardowski T. San Antonio J.D. J. Biol. Chem. 2000; 275: 7701-7707Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 35Fromm J.R. Hileman R.E. Caldwell E.E. Weiler J.M. Linhardt R.J. Arch. Biochem. Biophys. 1995; 323: 279-287Crossref PubMed Scopus (210) Google Scholar). For a similar reason, arginine polypeptide also binds with higher affinity to a negatively charged polymer like DNA. Thus, the stronger affinity for GAGs allows the more compact R16 complexes to accommodate low amounts of GAGs on the surface, leading to increase in size but minimal destabilization (Fig. 5A) along with increased transfection efficiency, whereas the loose packaging of K16 polyplexes is easily disturbed in presence of GAGs. It needs to be noted here that in terms of charge, 5 μg of HS has ∼5 times lesser negative charges than the positive charges on R16 peptides in a complex with DNA. Thus, if we assume that the GAGs attach to the complex surface, the polyplex still remains positively charged in the presence of the GAGs and possibly has a protective effect.We further dissected at which step the GAG protected R16 polyplexes score over the K16 polyplexes during cellular interaction. We observed that in the presence of GAGs at low concentrations (0.5:1 GAG:peptide) the R16 polyplex-GAG complex was associated with more than 95% of cells (at 4 °C) in the case of both the wild type and the glycan-deficient mutant cells as compared with only 30% cells when no GAG is present. This shows that the R16 polyplexes, in the presence of a GAG coating on them, show more cell association, but the binding sites possibly do not involve cell surface GAGs, as the effect is seen in both the wild type and the mutant cell line. Polyplexes were also internalized more efficiently in the presence of GAG coating both at 4 and 37 °C. Because internalization at 4 °C is usually indicative of a non-endocytotic route of entry, it is possible that multiple uptake routes are utilized by the GAG-coated R16 complexes. On the other hand, K16 polyplexes show decreased cell association and uptake in the presence of exogenous GAGs, which is even more prominent in the mutant cell line. Once again, this confirms the need for cell surface GAGs for the internalization of K16 polyplexes. But whether the cell surface proteoglycans are directly involved in the internalization of polyplexes by endocytosis or they act as primary receptors to present the complexes to specific endocytotic receptors is not clear and needs to be elucidated further.In summary, in this manuscript we have shown that arginine and lysine peptides have distinct behaviors in the presence of glycosaminoglycans. 16-Mer arginine homopeptides and their complexes with DNA can enter glycan-deficient cells as efficiently as the wild type cells, showing that cell surface GAGs are dispensable for their entry. But 16-mer lysine homopeptides and complexes prefer a GAG-dependent pathway for cellular entry. In the presence of relatively lower amounts of soluble GAGs, the arginine polyplexes show better stability and give an increase in the transfection efficiency in both cell types, unlike the lysine polyplexes. The chemical nature of the peptide and their different mechanisms of DNA compaction and release can thus cause differences in their behavior in the presence of both cell surface and soluble GAGs. The interaction between R16 and different GAGs does not seem to show specificity, as the consequential effects of R16 polyplex binding to GAGs remains similar for both HS and CS. The specificity might arise from differences in peptide length and structural characteristics as it has been demonstrated that the structures of arginine-rich peptides, specifically the distribution of positive charges, determine their dependence and specificity for heparan sulfate proteoglycan (17Nakase 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 (336) Google Scholar). In addition to stabilization of the complexes and their higher membrane binding affinity and uptake in the presence of GAGs, arginine polyplexes might also be adopting a different route of uptake or intracellular processing which delivers functional DNA more efficiently. This aspect is currently under investigation. We are also trying to explore whether conjugating GAG to peptide-DNA complexes will be useful in targeting specific cell types or whether this modulation will enhance transfection efficiency in a GAG-rich environment. IntroductionProteoglycans are biologically important molecules that are ubiquitously expressed on the cell surfaces as well as the extracellular and intracellular spaces and are involved in a variety of functions including tissue organization, morphogenesis, cellular communication, adhesion, migration, growth, and signaling to name few (1Bishop J.R. Schuksz M. Esko J.D. Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1239) Google Scholar). They are made up of a protein core linked to linear polysaccharide chains called glycosaminoglycans (GAGs). 3The abbreviations used are: GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; C6S, chondroitin 6-sulfate. These glycosaminoglycan chains are composed of different repeating disaccharide units that are sulfated at various positions. Based on the nature of the repeating units and the degree of sulfation, heparin, heparan sulfate, chondroitin sulfates, keratan sulfates, and hyaluronan are the different types of GAGs well known in the literature. Sulfated proteoglycans on the cell membranes are the major contributors to the negative charge on the cell surface, making it available for electrostatic interactions. These proteoglycans bind many ligands like growth factors and chemokines as well as large proteins. They have also been implicated in the internalization of foreign molecules and organisms through endocytotic pathways. Many viruses including adeno-associated virus (2Summerford C. Samulski R.J. J. Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar), herpes simplex virus, human papilloma virus, and human immunodeficiency virus (3Chen Y. Götte M. Liu J. Park P.W. Mol. Cells. 2008; 26: 415-426PubMed Google Scholar) initially bind to cell surface heparan sulfate proteoglycans to mediate their cellular entry.Non-viral gene delivery vectors like lipoplexes (lipid-DNA complexes) and polyplexes (polymer-DNA complexes), which are usually cationic in nature, can also interact with both cell surface and extracellular GAGs through electrostatic interactions. The cell surface GAGs have been thought to act as the primary receptor for the attachment of the cationic complexes followed by endocytosis of the complexes (for review, see Ref. 4Poon G.M. Gariépy J. Biochem. Soc. Trans. 2007; 35: 788-793Crossref PubMed Scopus (6) Google Scholar). Early reports showed that poly-l-lysine-DNA complexes as well as different cationic liposome-DNA complexes require cell surface proteoglycans (specifically, heparan sulfate proteoglycans) for delivery of DNA both in vitro and in vivo (5Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 6Mounkes L.C. Zhong W. Cipres-Palacin G. Heath T.D. Debs R.J. J. Biol. Chem. 1998; 273: 26164-26170Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). However, many subsequent reports have shown that their function in cellular entry of non-viral vector complexes may be dispensable, and strong binding of cationic complexes to either cell surface or extracellular GAGs may actually prevent their uptake. It was demonstrated that cellular uptake of cationic lipoplexes occurred at similar levels in cell lines with cell surface GAGs as well as in GAG-deficient cell lines (7Belting M. Petersson P. Biochem. J. 1999; 342: 281-286Crossref PubMed Scopus (39) Google Scholar), which implies that any role of GAGs in controlling transfection efficiency can at best come into play at a later step of the transfection process. It has also been suggested that cell surface proteoglycans protect cells from the cytotoxic effects of cationic lipids, and hence, proteoglycan-deficient cell lines give lowered transfection at high lipid to DNA ratios (7Belting M. Petersson P. Biochem. J. 1999; 342: 281-286Crossref PubMed Scopus (39) Google Scholar). In contrast, another study showed that cationic polymers such as poly-l-lysine and polyethyleneimine, and lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) and 1,2-dioleyl-3-phosphatidylethanolamine (DOPE) show strong binding to the cell surface GAGs but decreased transfection efficiency in their presence due to decreased cellular uptake (8Ruponen M. Honkakoski P. Tammi M. Urtti A. J. Gene Med. 2004; 6: 405-414Crossref PubMed Scopus (91) Google Scholar). The exact role of the cell surface proteoglycans in lipoplex or polyplex delivery is, thus, elusive, and different experimental conditions as well as different chemical and structural characteristics of the cationic carriers might be responsible for the differences observed in the data.Exogenous addition of negatively charged GAGs in vitro during transfection has also been studied in most of these systems and has usually been found to decrease the gene transfer efficiency (5Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 9Ruponen M. Rönkkö S. Honkakoski P. Pelkonen J. Tammi M. Urtti A. J. Biol. Chem. 2001; 276: 33875-33880Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). It is thought that depending on the chemistry of the carrier, the nature of the complex, and the charge density of GAGs, the decrease in transfection may be due to one or all of the following reasons. (i) Sulfated GAGs cause premature extracellular release of DNA from its complexes with cationic polymers (10Ruponen M. Ylä-Herttuala S. Urtti A. Biochim. Biophys. Acta. 1999; 1415: 331-341Crossref PubMed Scopus (305) Google Scholar, 11Burke R.S. Pun S. Bioconjugate Chem. 2008; 19: 693-704Crossref PubMed Scopus (170) Google Scholar) or cationic lipids (12Zelphati O. Szoka Jr., F.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11493-11498Crossref PubMed Scopus (589) Google Scholar). (ii) Soluble GAGs could compete with cell surface proteoglycans for binding to the complex, thereby decreasing cellular uptake (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). (iii) Binding of GAGs may change the endocytotic uptake route of the complex. (iv) GAGs may cause altered intracellular distribution of the complexes, significantly affecting the gene expression (9Ruponen M. Rönkkö S. Honkakoski P. Pelkonen J. Tammi M. Urtti A. J. Biol. Chem. 2001; 276: 33875-33880Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).The strong and universal translocation of arginine-rich cell-penetrating peptides in multiple cell types has also triggered speculation on the involvement of some common molecules like cell surface GAGs in the process of cellular entry, although it is still debatable whether membrane translocation and endocytotic uptake are both involved (14Futaki S. Nakase I. Tadokoro A. Takeuchi T. Jones A.T. Biochem. Soc. Trans. 2007; 35: 784-787Crossref PubMed Scopus (187) Google Scholar). Cell-penetrating peptides like naturally occurring protein transduction domains, e.g. the HIV-TAT peptide (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar), Antennapedia peptide (15Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), penetratin, and even synthetic homoarginines of certain compositions (16Fuchs S.M. Raines R.T. Biochemistry. 2004; 43: 2438-2444Crossref PubMed Scopus (320) Google Scholar, 17Nakase 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 (336) Google Scholar), are likely to involve cell surface GAGs in their cellular entry. It has also been seen that when these peptides are used for cargo delivery, the requirement of cell surface GAGs differs. Cellular uptake of HIV-1 TAT peptide conjugated to cargo was first surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans, which was confirmed by impaired uptake on enzymatic or genetic removal of the cell surface GAGs (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). However, uptake of free TAT peptide was suggested to involve either different receptors or pathways because its internalization was not completely inhibited in cells lacking surface heparan sulfate (19Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). The involvement of cell surface proteoglycans on the cellular entry of the peptide and its complexes with cargo can be affected by many factors such as net charge of the complex (18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) as well as the structure and distribution of positive charges on the peptides (17Nakase 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 (336) Google Scholar), and hence, it is possible that the bare peptide and the complex behave in different ways. More recently, it was proposed that transduction mediated by TAT can occur in glycan-deficient cell lines and the cell surface glycans bind TAT and only affect the efficiency of transduction (20Gump J.M. June R.K. Dowdy S.F. J. Biol. Chem. 2010; 285: 1500-1507Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, although arginine-rich peptides have high affinity to sulfated GAGs, especially heparan sulfate, and heparin (21Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and could co-internalize heparan sulfate, mediating nuclear delivery (18Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), soluble sulfated GAGs inhibited cargo delivery by arginine-rich peptides (13Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 15Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). All these evidences suggest that the role of GAGs in controlling cellular entry of cationic peptides with and without cargo needs further elucidation.Under this backdrop, we have explored the role of both cell surface and soluble GAGs during gene delivery using model peptide carriers, arginine, and lysine homopeptides (each 16-mer in length). Peptides containing these two amino acids either alone or in different proportions (e.g. TAT peptide) and often with simple chemical modifications like attachment of other additional amino-acids, polyethylene glycol, functional moieties like targeting ligands, and fusogenic peptides, etc. are the most commonly studied peptide based gene delivery agents (22Martin M.E. Rice K.G. AAPS J. 2007; 9: E18-E29Crossref PubMed Scopus (255) Google Scholar, 23Mann A. Thakur G. Shukla V. Ganguli M. Drug Discov. Today. 2008; 13: 152-160Crossref PubMed Scopus (56) Google Scholar). We show that the 16-mer arginine and lysine homopeptides differ in their requirement of cell surface GAGs as well as in their interaction with soluble GAGs during gene delivery. This is reflected in the alterations of complex morphology, pattern of cellular binding, internalization, and eventual gene delivery efficiency between the two peptide systems. Our results highlight the importance of peptide chemistry as a key determinant for requirement of GAGs in gene delivery." @default.
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• W2053873441 title "Exogenous and Cell Surface Glycosaminoglycans Alter DNA Delivery Efficiency of Arginine and Lysine Homopeptides in Distinctly Different Ways" @default.
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