Author manuscript, published in "The Journal of Biological Chemistry 2008;283(35):24274-84" DOI: 10.1074/jbc.M709971200 DIRECT PEPTIDE INTERACTION WITH SURFACE GLYCOSAMINOGLYCANS CONTRIBUTE TO THE CELL PENETRATION OF MAUROCALCINE

Maurocalcine (MCa), initially identified from a tunisian scorpion venom, defines a new member of the family of cell penetrating peptides (CPPs) by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a CPP implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans (HSPGs). Here, we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance (SPR). HP and HS bind both to MCa, indicating that HSPGs may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A, and (ii) the cell penetration of wild-type or mutant MCa, coupled to fluorescent streptavidin, is reduced by about 50% in mutant CHO cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP or chondroitin sulfates (CS), also inhibits the cell penetration of MCa/streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several CHO cell lines show that the distribution of the complex coincides with the endosomal marker lysotracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferin receptors, nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that: i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and ii) GAG-dependent and –independent MCa penetration rely similarly on the macropinocytosis pathway. Maurocalcine (MCa) is a 33-mer peptide isolated from the venom of the scorpion Scorpio maurus palmatus. MCa is a highly basic peptide since 12 out of 33 residues are positively charged including the amino terminal Gly residue, seven Lys residues and four Arg residues. Because it contains only four negatively charged residues, the net global charge of the peptide is also positive. MCa possesses three disulfide bridges connected according to the pattern Cys 3 –Cys 17 , Cys 10 –Cys 21 , and Cys 16 –Cys 32. 1 H-NMR analysis further indicates that MCa folds along an inhibitor cystine knot motif (1). MCa contains three β-strands running from amino acid residues 9–11 (strand 1), 20–23 (strand 2), …


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Maurocalcine (MCa) is a 33-mer peptide isolated from the venom of the scorpion
MCa is a highly basic peptide Scorpio maurus palmatus. inhibitor cystine knot motif ( ). MCa contains three -strands running from amino acid residues 9 11 (strand 1), 20 23 (strand 2), and 30 33 1 β ---(strand 3), respectively, with -strands 2 and 3 forming an antiparallel -sheet. MCa has proven to be a highly potent modulator of the skeletal β β muscle ryanodine receptor type 1 (RyR1), an intracellular calcium channel. Addition of MCa to the extracellular medium of cultured myotubes induces Ca release from the sarcoplasmic reticulum (SR) into the cytoplasm within seconds, as shown using a calcium-imaging approach ( , ). These observations, suggested that MCa is able to cross the plasma membrane to reach its pharmacological target. This was first demonstrated when a biotinylated analogue of MCa was coupled to a fluorescent derivative of streptavidin and the complex shown to cross the plasma membrane ( ). Cell penetration of this MCa-based complex is rapid, reaches saturation within minutes, and occurs at concentrations as 4 low as 10 nM ( ). Furthermore, an alanine scan of MCa indicates the importance of basic amino acid residues in the cell penetration 5 mechanism. Reducing the net positive charge of the molecule appears to decrease its cell penetration efficiency. In parallel, MCa analogues exhibiting decreased penetration efficiency were also found to present reduced affinity for negatively charged lipids of the plasma membrane (6 ).
Over the past years, several peptides have been characterized for their ability to cross the plasma membrane ( ). Cell penetration of 7-11 peptides obeys three fundamental steps: (i) binding to some components of the plasma membrane, (ii) the cell entry process , and (iii) the per se subsequent release into the cytoplasm. Obviously, none of these steps are well understood and conflicting reports have emerged that may well arise from differences in the nature of the cell penetrating peptide (CPP) considered, cell preparations, experimental conditions, type of linkage to cargoes, or even cargo nature. Two non-competing mechanisms have been proposed for the cell entry of CPP. One is direct translocation through the plasma membrane by the CPP-induced reorganization of the membrane following several possible structural alterations ( ). [7][8][9][10][11] According to some investigators, this mechanism implies a direct CPP interaction with negatively charged lipids of the plasma membrane. This mechanism of penetration would be independent of both cell metabolic energy and membrane receptor presence. For instance, it was proposed that penetratin binds to the polar heads of lipids leading to the formation of inverted micelles, followed by a subsequent opening of these micelles inside the cell and the release of the peptide into the cytoplasm ( ). A second mechanism involves a form of endocytosis by which 12 the CPP gets localized into late endosomes from where it may eventually leak out partially towards the cytoplasm. Endocytosis can be initiated by binding of CPPs to HS along with binding to negatively charged moieties on the cell surface, such as lipids ( ). Lipid-raft dependent 13 macropinocytosis has been evidenced as one endocytosis pathway for the cell entry of CPPs ( , ). For instance, cellular uptake of a 14 15 recombinant GST-TAT-GFP fusion protein depends on the presence of HS proteoglycans (HSPG) at the cell surface ( ). Nevertheless, the 16 role of GAGs in the cell penetration of CPPs remains debated. Stereochemistry, chain length, patterns of sulfation and negative charge distribution of GAGs lead to a great variety of protein binding motifs. Furthermore, the CPP structure also appears to determine its specificity for HSPG ( ). 17 In the present study, we show that MCa interacts with GAGs such as HS and HP with apparent affinities in the micromolar range. A less penetrating analogue of MCa (MCa K20A) also shows a reduced apparent affinity for these GAGs suggesting a direct link between GAG interaction and cell penetration. Cell penetration of MCa -streptavidin complex is strongly inhibited by an inhibitor of macropinocytosis b indicating that this route of entry is responsible for MCa penetration. However, use of GAG-deficient cell lines indicates that half of the cell penetration of the complex is conserved and still relies on macropinocytosis. We conclude that GAG-dependent and independent entries of -MCa use similar pathways. Cell surface GAGs appear important to specify a higher cell penetration level, but penetration still can occur in their absence presumably because binding onto lipids can also activate macropinocytosis.

Equipment and reagents
The Biacore 3000 apparatus, CM4 sensor chips, amine coupling kit and HBS-P buffer (   Incubation of the membranes with 100 nM biotin alone was used as a negative control condition. The membranes were then washed a first time with TBS-T 0.1 free BSA using a gentle agitation for 10 min. In all conditions, MCa or biotin binding onto the lipid spots was detected by a % b 30 min incubation with 1 g/ml streptavidin horse radish peroxidase (Vector labs, SA-5704), followed by a second wash with TBS-T 0.1 free μ % BSA, and an incubation with horse radish peroxidase substrate (Western Lightning, Perkin-Elmer Life Science) for 1 min. Lipid membranes were then exposed to a Biomax film (Kodak). The intensity of interaction with the lipids was analyzed with Image J (National Institute of Health, USA).

MCa interacts with HS and HP
Pre-incubation of MCa with HP was found to partially inhibit its penetration in HEK293 cells ( ). To evaluate the binding of MCa to b 5 HSPGs, HP or HS were coupled to a Biacore sensorchip and the MCa binding monitored by SPR ( ). Injection of a range of MCa Figure 1 concentrations (up to 5 M) over HP-or HS-coupled sensorchips gave rise to increasing binding amplitudes as shown in . A mutated μ Figure 1B analogue of MCa (MCa K20A) showed impaired binding activity, indicating the importance of residue K20 for glycosaminoglycan recognition.
This finding is in agreement with the role of HSPGs in the cell entry of CPPs and with the observation that the MCa K20A has impaired cell penetration ( ). The data could not be fitted to a binding model, presumably because all binding curves had a square shape with sharp edges, 6 " " suggesting high association and dissociation rates. We were thus not able to extract reliable kinetic values from curve fitting. Equilibrium data, plotted according to the Scatchard representation, were used to determine affinity ( ). The straight lines obtained show that MCa Figure 1C  HP is formed by the polymerization of a various number of disaccharide units. In order to study the effect of the size of the polymer on its interaction with MCa, we performed competition experiments using HP-derived oligosaccharides of defined degree of polymerization (dp) ( ). For this purpose, wild-type MCa was pre-incubated with different HP-derived oligosaccharides (dp6, dp12 or dp18), as mentioned Figure 1D in the , and then injected over the HP-conjugated sensorchip. As shown, the oligosaccharides caused a dose-dependent Materials and Methods inhibition of the interaction of MCa with HP. dp18 was the most active oligosaccharide with an IC close to 1 M. In contrast, dp6 had almost 50 μ no effect at 5 M.  . In wild-type CHO cells, HS (250 g/ml) produced the most potent inhibition of cell penetration (84 2 , n 3, ). HP and Figure 3 μ ± % = Figure 3A CS were less efficient than HS with a mean inhibition of 59 10 (n 3) and 19 2 (n 3) for HP and CS, respectively (at 250 g/ml).

MCa -Strep-Cy5 localizes to endosomal structures that do not originate from clathrin-mediated endocytosis b
Using confocal microscopy, the cell distribution of MCa -Strep-Cy5 was compared to that of endosomal structures as revealed by b lysotracker red staining. As shown on there is a very good co-localization between MCa -Strep-Cy5 and lysotracker red fluorescence  Expression of a dominant-negative mutant of dynamin 2, dynamin 2 K44A, is known to prevent normal clathrin-mediated endocytosis ( ). 26 As shown in , expression of dynamin 2 K44A prevents the entry of transferrin-alexafluor-594 in both wild-type and GAG-deficient Figure 7A CHO cells confirming that transferrin receptors get internalized by clathrin-mediated endocytosis. In contrast, MCa -Strep-Cy3 entry was not b prevented by the expression of dynamin 2 K44A ( ), clearly indicating that clathrin-mediated endocytosis was not required for the Figure 7B entry of MCa when coupled to streptavidin. b

Lack of alteration of the main endocytosis entry pathway in GAG-depleted cells
To   on Strep-Cy3. The exact molar ratio between MCa and Strep-Cy3 can however not be warranted by simply mixing various molar ratios of the b two molecules. Once these complexes were prepared, their cell entry, along with the effect of amiloride, were quantified by FACS ( ). Figure 9 Increasing the molar ratio of MCa over Strep-Cy3 from 1:1 to 8:1 dramatically increases the amount of Strep-Cy3 that penetrates into b wild-type CHO cells ( ). These data indicate that immobilizing an increasing number of MCa onto streptavidin greatly favors the Figure 9A b entry of the complex, possibly by multiplying the number of contacts with cell surface components and/or increasing the residency time at the cell surface. In contrast, using increased amounts of non-biotinylated MCa, unable to bind Strep-Cy3, in place of MCa , did not produce any b increase in Strep-Cy3 penetration indicating that coupling of MCa to Strep-Cy3 was required ( ). This result also shows that the Figure 9B association

Macropinocytosis is the main endocytic pathway used by MCa when coupled to streptavidin in GAG-positive and GAG-deficient CHO cells
In a previous work, we reported that MCa-Strep complex penetration in HEK 293 cells was also observed in the presence of amiloride or nystatin suggesting that a non-endocytic pathway was involved in the penetration process ( ). Here, we provide a quantitative analysis of the of effect of methyl--cyclodextrin appears to indicate that endocytosis of MCa -Strep-Cy5 is not dependent on lipid rafts or at least on β b cholesterol availability. Since macropinocytosis appears to be responsible for the uptake of MCa -Strep-Cy5/3 in wild-type and GAG-deficient b CHO cells alike, it seems that all surface components able to bind MCa, negatively-charged HSPGs and lipids, are involved in macropinocytosis. Owing to the nature of macropinosomes, that do not fuse with lysosomes and that are leaky, it is likely that release of CPPs in the cytosol may occur very slowly. In the case of the Strep-Cy5 cargo, this leakage was however not observed when coupled to MCa .
b Cargo-dependence of MCa mode of penetration and/or release in the cytosol?
The mechanism of cell penetration of CPPs remains highly debated. There are pro and con arguments in favor of membrane translocation, a process whereby the peptide would flip from the outer face of the plasma membrane to the inner face, then released free into the cytoplasm.
Here, we do not provide compelling evidence for a translocation mechanism for Strep-Cy5 entry when coupled to MCa . On the contrary, the cargo may itself represent a problem for simple diffusion of the complex from leaky macropinosomes to the cytosol. Further studies will be " " required to investigate the contribution of cargo size and nature in the mode of entry and cell distribution (cytosol endosomes) of the versus vector. Nevertheless, these data are coherent with many other studies on CPPs and macropinocytosis is likely to be the main entry route of many other cargoes that will be attached to maurocalcine. Although streptavidin is used as a reporter cargo here (fluorescence property), it is worth mentioning that its ability to bind to several different biotinylated molecules at a time should be considered as a significant advantage for the cell delivery of multiple cargoes with a single MCa vector. = are normalized with mean fluorescence intensity of wild-type CHO cells.

Fig. 3
Inhibition of MCa -Strep-Cy5 cell penetration by soluble GAGs in wild-type and GAG-deficient CHO cell lines. (A) Dose-dependent inhibition of