Guanidinylated Neomycin Mediates Heparan Sulfate–dependent Transport of Active Enzymes to Lysosomes
Authors: Stéphane Sarrazin, Beth Wilson, William S Sly, Yitzhak Tor, Jeffrey D Esko
Journal: Molecular Therapy (2010)

Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, β-d-glucuronidase (GUS) and α-l-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells. Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, β-d-glucuronidase (GUS) and α-l-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells. IntroductionHeparan sulfate proteoglycans are abundantly expressed on virtually all animal cells and bind a large number of ligands, including growth factors and morphogens, proteases and their inhibitors, as well as lipoproteins and the lipases that act on them.1Bernfield M Götte M Park PW Reizes O Fitzgerald ML Lincecum J et al.Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2295) Google Scholar,2Bishop JR Schuksz M Esko JD Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1241) Google Scholar,3Bishop JR Stanford KI Esko JD Heparan sulfate proteoglycans and triglyceride-rich lipoprotein metabolism.Curr Opin Lipidol. 2008; 19: 307-313Crossref PubMed Scopus (37) Google Scholar Proteoglycans undergo constitutive endocytosis, apparently through a clathrin-independent pathway,4Wilsie LC Gonzales AM Orlando RA Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway.Lipids Health Dis. 2006; 5: 23Crossref PubMed Scopus (39) Google Scholar and eventually arrive at the lysosome.5Fuki IV Meyer ME Williams KJ Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts.Biochem J. 2000; 351: 607-612Crossref PubMed Google Scholar,6Burbach BJ Friedl A Mundhenke C Rapraeger AC Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells.Matrix Biol. 2003; 22: 163-177Crossref PubMed Scopus (51) Google Scholar Various lysosomal proteases, sulfatases, and glycosidases subsequently degrade the proteoglycan core protein and the heparan sulfate chains.7Winchester BG Lysosomal metabolism of glycoconjugates.Subcell Biochem. 1996; 27: 191-238Crossref PubMed Scopus (37) Google Scholar,8Bame KJ Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans.Glycobiology. 2001; 11: 91R-98RCrossref PubMed Scopus (136) Google Scholar Ligands bound to the chains also undergo degradation. For example, the heparan sulfate proteoglycan syndecan-1 expressed by hepatocytes binds triglyceride-rich remnant lipoproteins, resulting in their internalization and degradation in the liver. Loss of syndecan-1 expression results in hypertriglyceridemia, demonstrating at least one physiological role for this uptake system.9Stanford KI Bishop JR Niesman IR Witztum JL Esko JD Syndecan-1 is the primary heparan sulfate proteoglycan mediating hepatic clearance of triglyceride-rich lipoproteins in vivo.J Clin Invest. 2009; 119: 3236-3245PubMed Google ScholarGuanidinoglycosides are derivatives of the naturally occurring aminoglycoside antibiotics in which all the ammonium groups have been converted into guanidinium groups.10Luedtke NW Baker TJ Goodman M Tor Y Guanidinoglycosides: a novel family of RNA ligands.J Am Chem Soc. 2000; 122: 12035-12036Crossref Scopus (107) Google Scholar,11Luedtke NW Carmichael P Tor Y Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine.J Am Chem Soc. 2003; 125: 12374-12375Crossref PubMed Scopus (131) Google Scholar For example, guanidinylated neomycin (GNeo) contains six positively charged guanidinium groups in place of the naturally occurring amino groups on the four monosaccharide units that make up the antibiotic. Noncovalent attachment of GNeo to large bioactive molecules (>300 kd) by way of biotin–streptavidin interaction facilitates their transit across cell membranes.12Elson-Schwab L Garner OB Schuksz M Crawford BE Esko JD Tor Y Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.J Biol Chem. 2007; 282: 13585-13591Crossref PubMed Scopus (64) Google Scholar At low nanomolar transporter concentrations, these carriers deliver their cargo in a heparan sulfate exclusive manner based on the refractory behavior of mutant cells lacking heparan sulfate. Conjugation of GNeo to the fluorophores phycoerythrin-Cy5 and Alexa488 demonstrated uptake into subcellular organellar compartments. A GNeo conjugate containing saporin, a ribosome inactivating protein, apparently can gain access to the cytoplasm and kill the cells in a heparan sulfate–dependent manner. Whether protein ligands containing GNeo continue to progress through the vesicular system in cells to other subcellular organelles was unclear.In this report, we demonstrate that GNeo, conjugated to quantum dots via biotin–streptavidin, enables particle entry into cells in a heparan sulfate–dependent manner and facilitates their lysosomal accumulation. We also describe an N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS), which facilitates covalent conjugation under mild conditions of the transporter to lysine residues exposed on the surface of proteins. Conjugation of GNeo to β-d-glucuronidase (GUS) and α-l-iduronidase in this fashion does not interfere with enzymatic activity, endows the enzymes with the capacity to bind heparin, and enables their delivery to lysosomes. Sufficient amounts of enzymes are delivered to restore the turnover of glycosaminoglycans in cells lacking endogenous forms of these enzymes. We conclude that cell surface heparan sulfate proteoglycans have sufficient capacity to deliver therapeutic doses of enzymes, opening up the possibility of using guanidinylated glycosides as a general vehicle for enzyme delivery.ResultsGNeo-conjugated quantum dots bind to heparin and heparan sulfate, and appear in lysosomesIn previous studies, we conjugated biotinylated GNeo to Streptavidin-Alexa488 to monitor cell binding and uptake by fluorescence microscopy. Labeling studies of Chinese hamster ovary (CHO) cells showed initial uptake of the fluorophore into vesicular structures. Uptake depended on the presence of cell surface proteoglycans based on the absence of signal in mutant cells unable to make glycosaminoglycans (pgsA).12Elson-Schwab L Garner OB Schuksz M Crawford BE Esko JD Tor Y Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.J Biol Chem. 2007; 282: 13585-13591Crossref PubMed Scopus (64) Google Scholar In a similar way, we conjugated biotinylated GNeo to streptavidin-coated quantum dots (QD525), which are more photostable and resistant to degradation compared to conventional fluorophores. As a result, many consecutive focal-plane images can be reconstructed into a high-resolution three-dimensional image, and real-time tracking in cells can be achieved over extended periods of time. Each quantum dot contains from 5–10 molecules of streptavidin each containing four binding sites for biotin. Pilot experiments demonstrated that extensive binding to heparin-Sepharose occurred when the biotin acceptor sites were saturated with GNeo. GNeo-QD525 prepared in this way bound strongly to heparin-Sepharose and eluted between 0.9 and 1.8 mol/l NaCl (data not shown). Conjugation did not affect the excitation or emission spectra (data not shown).Incubation of wild-type CHO cells with 5 nmol/l GNeo-QD525 led to fluorescent labeling of the cells, which was easily quantified by flow cytometry (Figure 1a, black line). Significant fluorescence was observed compared to controls in which the quantum dots were not added to the cells (shaded area in Figure 1a) or in cells incubated with quantum dots not derivatized with GNeo (QD525, gray line). Under these conditions, the majority of the fluorescence signal was due to uptake of GNeo-QD525 because the cells were treated with trypsin prior to flow cytometry. Control experiments in which cells were incubated with GNeo-QD525 at 4 °C showed that nearly all of the cell-associated material was released by trypsin treatment at low temperature (Supplementary Figure S1). In contrast, at 37 °C about 20% of GNeo-QD525 was resistant to trypsin treatment, a value that increased with time. Uptake was completely dependent on glycosaminoglycans because pgsA-745 cells lacking both heparan sulfate and chondroitin sulfate did not show a significant signal by flow cytometry (Figure 1b). Similar results were obtained in pgsD-677 cells, which lack heparan sulfate and make about two- to threefold more chondroitin/dermatan sulfate (Figure 1c). Thus, GNeo-conjugated quantum dots behaved much like GNeo-streptavidin-Alexa488 and GNeo-streptavidin-phycoerythrin-Cy5 conjugates described previously in terms of their high selectivity for heparan sulfate.12Elson-Schwab L Garner OB Schuksz M Crawford BE Esko JD Tor Y Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.J Biol Chem. 2007; 282: 13585-13591Crossref PubMed Scopus (64) Google ScholarImaging of the cells by deconvolution fluorescence microscopy showed that GNeo-QD525 was present in punctate structures (Figure 2a, green). Uptake was a relatively slow process because GNeo-QD525 did not appear inside the cells for ∼45 minutes (data not shown). Many of the punctate structures containing GNeo-QD525 at 37 °C also co-stained with LysoTracker (red) (Figure 2b,c). A three-dimensional volumetric rendering and fly-through is shown in the Supplementary Video S1, which demonstrated that the majority (∼90%) of internalized GNeo-conjugated quantum dots colocalized with lysosomes after 3 hours. These findings indicated that GNeo could deliver very high molecular weight cargo (estimated size of the streptavidinylated quantum dots >107 Da) to lysosomes by way of cell surface heparan sulfate proteoglycans.Figure 2GNeo-QD525 colocalize in lysosomes. Wild-type Chinese hamster ovary cells were incubated with 5 nmol/l GNeo-QD525 in growth medium for 30 minutes. After rinsing the cells three times, fresh medium was added, and 2.5 hours later, they were rinsed with Hank's balanced salt solution and labeled with Hoechst dye and LysoTracker Red. Images were captured with a DeltaVision Restoration microscope system and were deconvolved to show the localization of (a) GNeo-QD525, (b) lysosomes in a single Z-stack plane. The merged images from a and b are shown in c with the outline of cells (hatched line) drawn based on a phase contrast micrograph. Bar = 5 µm. A three-dimensional reconstruction is shown in Supplementary Video S1 to better appreciate the extensive colocalization of quantum dots with the lysosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Conjugation of GNeo to enzymes confers heparin bindingThe discovery that GNeo-conjugated quantum dots appeared in lysosomes suggested the use of GNeo to deliver lysosomal enzymes to cells. Initial discussions with W.S.S. (St Louis University, St Louis, MO) and Elizabeth Neufeld (University of California–Los Angeles, Los Angeles, CA) suggested that GUS was an ideal enzyme to initiate these studies due to the availability of the purified bovine and recombinant human enzymes, its relative stability compared to other lysosomal enzymes, and the availability of human fibroblasts from mucopolysaccharidosis (MPS) VII patients (Sly's syndrome) lacking endogenous GUS.To attach GNeo to the enzyme, a linker containing a terminal NHS-activated ester was conjugated to GNeo (GNeo-NHS, Figure 3). A key step in fabricating this relatively reactive derivative is a 2+3 cycloaddition reaction (commonly referred to as a Click reaction) between an NHS-activated azido carboxylic acid and an alkyne-linked guanidinoglycoside derivative (see Supplementary Scheme S1 and Supplementary Materials and Methods for synthetic procedures and analytical data). The necessary building blocks, including the singly modified neomycin core, were prepared according to previously published procedures.13Baker TJ Luedtke NW Tor Y Goodman M Synthesis and anti-HIV activity of guanidinoglycosides.J Org Chem. 2000; 65: 9054-9058Crossref PubMed Scopus (99) Google Scholar,14Kirk SR Luedtke NW Tor Y Neomycin–acridine conjugate: a potent inhibitor of Rev-RRE binding.J Am Chem Soc. 2000; 122: 980-981Crossref Scopus (142) Google Scholar Enzyme conjugates were prepared by reacting bovine GUS (bGUS) with GNeo-NHS at various ratios (e.g., ten-, 50-, and 100-fold molar excess of the NHS derivative). A high molar excess (>50-fold) was necessary to achieve conjugation levels that enabled binding to heparin-Sepharose. Under these conditions, about two-thirds of the treated enzyme did not bind to the resin, like native bGUS. The remainder required 0.3–0.9 mol/l NaCl to elute from the resin (Supplementary Figure S2a). The addition of GNeo had at most only a minor effect on enzyme-specific activity (∼30% in the unfractionated preparation, but no effect on the modified enzyme that eluted at 0.3 and 0.6 mol/l NaCl; Supplementary Figure S2b).Figure 3Generation of GNeo-N-hydroxysuccinimide (GNeo-NHS) by "Click" chemistry.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Uptake of GNeo-GUS by cells depends on heparan sulfateTo test whether GNeo-bGUS was taken up by GUS-deficient cells, enzyme activity was measured in MPS VII fibroblasts after 2-hour incubation with various concentrations of unmodified bGUS and GNeo-bGUS (Figure 4a). Poor uptake of bGUS occurred at all concentrations tested, whereas uptake of GNeo-bGUS was proportional to concentration up to 450 nmol/l, the highest concentration tested. When cells were incubated with 20 nmol/l of GNeo-bGUS for 2 hours, the cells took up 0.5% of the enzyme, whereas in the absence of GNeo conjugation, cells took up only 0.06% of the added enzyme. Neither bGUS nor GNeo-bGUS was degraded in cell-free extracts based on enzymatic activity assays, suggesting that incorporated enzyme was stable.Figure 4GNeo delivery of GUS is M6P-independent. (a) MPS VII fibroblasts were treated with the indicated concentration of bGUS or GNeo-bGUS for 2 hours. The cells were washed, trypsin treated, sedimented by centrifugation, washed three times and subsequently assayed for β-glucuronidase activity. (b) MPS VII cells were treated with 5 nmol/l of the indicated bovine enzyme preparations in growth medium in the absence (open bars) or in the presence of 5 mmol/l mannose-6-phosphate (filled bars) or glucose-6-phosphate (gray bars), and assayed for β-glucuronidase activity. Assays of normal human foreskin fibroblasts (HFF) and MPS VII cells are shown for comparison. The difference between bGUS uptake in the presence of M6P was significant, whereas the inhibition by G6P was not. Analysis of variance showed that these differences were significant (P = 0.0104). The differences within the other data sets were not significant (P = 0.2915 for HFF cells, P = 0.8595 for GNeo-bGUS, P = 0.4577 for AP-bGUS, and P = 0.4513 for GNeo-AP-bGUS). (c) MPS VII cells were treated with 1 nmol/l of the indicated human enzymes in the presence (filled bars) and absence (open bars) of 5 mmol/l mannose-6-phosphate and assayed for β-glucuronidase activity. Assays of normal human fibroblasts (HFF) and untreated MPS VII cells are shown for comparison. (d) MPS VII cells were treated with medium in the absence (open bars) or with a mixture of heparin lyases (filled bars) 15 minutes prior to addition of 5 nmol/l bGUS or GNeo-bGUS or 1 nmol/l of hGUS or GNeo-AP-hGUS. Error bars are the SEM of triplicate uptake experiments. The difference between GNeo-bGUS uptake after heparinase digestion and bGUS was significant (P = 0.009) and probably reflected incomplete digestion by heparinases. The difference in hGUS uptake after heparinase was not significant (P = 0.2). The data were compared by Student's t-test. G6P, glucose 6-phosphate; M6P, mannose-6-phosphate; MPS, mucopolysaccharidosis; RFU, relative fluorescence units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lysosomal enzymes are normally sorted to lysosomes by way of receptors that recognize mannose-6-phosphate (M6P)-terminated, asparagine-linked glycans.15Kaplan A Achord DT Sly WS Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.Proc Natl Acad Sci USA. 1977; 74: 2026-2030Crossref PubMed Scopus (381) Google Scholar,16Kornfeld S Trafficking of lysosomal enzymes.FASEB J. 1987; 1: 462-468Crossref PubMed Scopus (345) Google Scholar,17Ghosh P Dahms NM Kornfeld S Mannose 6-phosphate receptors: new twists in the tale.Nat Rev Mol Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (774) Google Scholar A portion of the cation-independent M6P receptors (CI-MPR) appears at the cell surface and mediates the uptake of "high-uptake" forms of enzymes that contain the M6P-terminated glycans. High concentrations of M6P (5 mmol/l) block this uptake route, whereas glucose 6-phosphate (G6P) does not. As shown in Figure 4b, endogenous GUS activity in human foreskin fibroblasts (HFF) was not affected by M6P or G6P. Uptake of bGUS was reduced by 50%, however, by 5 mmol/l M6P but not by G6P. Analysis of variance showed that these differences were significant (P = 0.0104). Treatment of bGUS with alkaline phosphatase (AP), which removes the terminal 6-phosphate group necessary for recognition, reduced uptake by ∼50% as well, and the residual uptake was insensitive to M6P, consistent with the presence of a M6P-independent receptor on fibroblasts.18González-Noriega A Michalak C Mannose 6-phosphate-independent endocytosis of beta-glucuronidase. II. Purification of a cation-dependent receptor from bovine liver.Biochim Biophys Acta. 2001; 1538: 152-161Crossref PubMed Scopus (11) Google Scholar In comparison, uptake of human recombinant GUS (hGUS) was robust (Figure 4c), and the addition of M6P or treatment with AP-hGUS greatly inhibited uptake.To test whether GNeo could confer high-uptake properties to GUS isoforms, conjugates of bGUS, AP-bGUS, or AP-hGUS were generated and added to human fibroblasts. The addition of GNeo dramatically increased enzyme uptake compared to the unmodified enzymes, exceeding the endogenous activity observed in untreated cells (Figure 4b,c). Importantly, free M6P had little, if any, effect on uptake, suggesting that the conjugated enzymes were not internalized via the CI-MPR pathway. Instead, uptake of the GNeo-conjugated enzymes depended on heparan sulfate, based on loss of uptake by prior treatment of the cells with heparin lyases, which depolymerizes the heparan sulfate chains on the surface of the cell. The incomplete inhibition of uptake of GNeo-bGUS by heparinase to the level observed with unmodified bGUS probably reflects incomplete digestion of heparan sulfate in this experiment and the presence of M6P-modified enzyme. The greater sensitivity of GNeo-AP-hGUS to heparinase probably reflects the lack of any M6P targeting signals in this preparation and the lower concentration of enzyme compared to bGUS (1 nmol/l versus 5 nmol/l, respectively). Uptake of unconjugated bGUS and hGUS was insensitive to treatment with heparin lyases.Internalized enzymes restore normal GAG turnoverTo test whether internalized GUS was functionally localized in lysosomes, we utilized a label-chase format in which cells were incubated with 35S-labeled sulfate for 24 hours to radiolabel the sulfated glycosaminoglycans. The medium was changed, and after 24 hours, the amount of [35S]glycosaminoglycans that remained associated with the cells was quantitated. Under these conditions, MPS VII fibroblasts retained about tenfold more [35S]glycosaminoglycans than normal HFF (Figure 5a).Figure 5GNeo delivery of lysosomal enzymes enhances turnover of glycosaminoglycans in mucopolysaccharidosis (MPS) fibroblasts. (a,b) Normal and MPS VII or (c) MPS I fibroblasts were radiolabeled with 35SO4 and chased for 24 hours with the indicated concentration of bovine β-glucuronidases (a), human β-glucuronidases (b), and human α-l-iduronidase (c). The amount of [35S]glycosaminoglycan remaining was measured (Materials and Methods). (a) GUS (squares), AP-GUS (triangles), or GNeo-AP-GUS (filled circles). (b) hGUS (squares), AP-hGUS (triangles), or GNeo-AP-GUS (filled circles). (c) Iduronidase (squares), AP-iduronidase (triangles), or GNeo-AP-iduronidase (filled circles). The dotted line represents the amount of [35S]glycosaminoglycan remaining in normal fibroblasts (HFF) without enzyme supplementation. The experiment was performed twice, in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Incubation of the cells with GNeo-AP-bGUS induced turnover, with an ED50 value of 150 mU of enzyme activity (Figure 5a). bGUS and AP-bGUS also enhanced the turnover of the [35S]glycosaminoglycans, but the ED50 values were tenfold higher, ∼1,500 mU (Figure 5a). The uptake mechanism of AP-bGUS has not been well characterized, and may involve other receptors or fluid-phase pinocytosis.18González-Noriega A Michalak C Mannose 6-phosphate-independent endocytosis of beta-glucuronidase. II. Purification of a cation-dependent receptor from bovine liver.Biochim Biophys Acta. 2001; 1538: 152-161Crossref PubMed Scopus (11) Google Scholar Recombinant hGUS, which is extensively modified with M6P, stimulated [35S]glycosaminoglycans turnover with a low ED50 (∼3 mU) and treatment with AP reduced its potency (ED50 ∼300 mU) (Figure 5b). The addition of GNeo to AP-hGUS restored its efficacy to a level comparable to hGUS (ED50 ∼10 mU) and with a similar dose–response curve.To demonstrate the general utility of GNeo as a transporter, we applied the same coupling method to α-l-iduronidase, a lysosomal enzyme missing in MPS I patients (Hurler, Hurler–Scheie, and Scheie syndromes). Like MPS VII cells, MPS I fibroblasts also stored [35S]glycosaminoglycans compared to wild-type HFF (Figure 5c). As expected, recombinant therapeutic α-l-iduronidase (Aldurazyme) restored turnover, whereas AP-α l iduronidase was comparatively ineffective (ED50 = 1 U versus 30 U, respectively). Conjugating GNeo to AP-α-l-iduronidase enhanced its uptake, shifting the ED50 to 0.2 units, making it as effective or better than native Aldurazyme in restoring [35S]glycosaminoglycan turnover.DiscussionTremendous progress has been made in recent years in the application of arginine-rich protein transduction domains (also named cell-penetrating peptides) in either chimerically expressed recombinant proteins or as tags for cellular delivery.19Tilstra J Rehman KK Hennon T Plevy SE Clemens P Robbins PD Protein transduction: identification, characterization and optimization.Biochem Soc Trans. 2007; 35: 811-815Crossref PubMed Scopus (34) Google Scholar,20Vives E Cellular uptake [correction of utake] of the Tat peptide: an endocytosis mechanism following ionic interactions.J Mol Recognit. 2003; 16: 265-271Crossref PubMed Scopus (147) Google Scholar,21Futaki S Oligoarginine vectors for intracellular delivery: design and cellular-uptake mechanisms.Biopolymers. 2006; 84: 241-249Crossref PubMed Scopus (163) Google Scholar The intricacies of entry, localization, and release of these peptide-based transporters remain somewhat controversial.22Foerg C Merkle HP On the biomedical promise of cell penetrating peptides: limits versus prospects.J Pharm Sci. 2008; 97: 144-162Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Multiple uptake pathways are likely to operate simultaneously, and their relative significance might depend on the specific sequences and cell types used.23Nakase I Takeuchi T Tanaka G Futaki S Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides.Adv Drug Deliv Rev. 2008; 60: 598-607Crossref PubMed Scopus (300) Google Scholar Compared to arginine-rich transduction peptides, guanidinoglycoside-based transporters, a family of synthetic derivatives where all the ammonium groups of aminoglycosides have been converted into guanidinium groups, display a unique entry pathway. At low carrier concentrations (nanomolar range), a high selectivity for cell surface heparan sulfate proteoglycans was observed. This finding suggested that guanidinoglycosides were likely to enter the cell complexed to glycosaminoglycan chains and immediately sparked the hypothesis that such carriers could effectively deliver high molecular weight cargo to organelles where heparan sulfate was stored and metabolized.12Elson-Schwab L Garner OB Schuksz M Crawford BE Esko JD Tor Y Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.J Biol Chem. 2007; 282: 13585-13591Crossref PubMed Scopus (64) Google ScholarTo experimentally investigate this hypothesis and to provide a proof of concept, we showed that GNeo-conjugation to quantum dots enabled the uptake of this very high molecular weight cargo (>107 Da) in a heparan sulfate–dependent manner (Figure 1). Although the specific vesicular route of uptake remains to be determined, uptake occurred relatively slowly with a t1/2 of >30 minutes, consistent with other studies suggesting that proteoglycans are internalized in a clathrin-independent manner, possibly dependent on lipid rafts and/or macropinocytosis.4Wilsie LC Gonzales AM Orlando RA Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway.Lipids Health Dis. 2006; 5: 23Crossref PubMed Scopus (39) Google Scholar Further studies are underway to identify the relevant proteoglycan(s) and their mechanism of internalization.Regardless of the mechanism of uptake, internalization led to the colocalization of the quantum dots with LysoTracker Red, suggesting delivery to lysosomes. To prove that the GNeo carrier was capable of delivering bioactive cargo, we developed an effective conjugation method for proteins using an activated ester derivative (GNeo-NHS). We selected GUS as the cargo due to its availability and stability, and because cell lines lacking the enzyme have been derived from deficient patients. The enzyme exists as a 300 kd tetramer and contains 27 lysine residues per monomer.24Oshima A Kyle JW Miller RD Hoffmann JW Powell PP Grubb JH et al.Cloning, sequencing, and expression of cDNA for human beta-glucuronidase.Proc Natl Acad Sci USA. 1987; 84: 685-689Crossref PubMed Scopus (145) Google Scholar An X-ray crystal structure shows that many of the lysines are accessible on the protein surface (PDB structure: 1BHG). It is unclear how many of these residues underwent modification by GNeo-NHS, but in other data not reported here, we have shown that GNeo by itself binds avidly to heparin (L. Fischer, A. Dix, S. Sarrazin, J. Esko, and Y. Tor, unpublished results). Thus, purification of the GUS-GNeo conjugates was facilitated by "affinity" chromatography on heparin-Sepharose (Supplementary Figure S2). Rewardingly, the GNeo-conjugated enzyme was found to maintain its hydrolytic activity as determined by in vitro fluorescence-based assays. In addition, the enzyme conjugates were found to retain their activity upon exposure to cells and cell extracts. Future studies will address the question of how binding and uptake are affected by the extent of modification by GNeo, which might be best approached using synthetic scaffolds where the number of GNeo moieties can be controlled.Normal routing of newly made lysosomal enzymes to lysosomes occurs in sorting vesicles in the interior of the cell