Polymerization of CaATP-G-actin by histatin-3 and histatin-5
Divalent and polyvalent cations polymerize G-actin to F-actin. The efficiency of polycations as polymerizing agents increases with the number of their net positive charges. We studied the kinetics and extent of actin polymerization by histatin-3 and histatin-5 (Fig. 1). The speed and extent of polymerization was histatin-concentration dependent. However, a considerable difference was found in the ability of the two histatins to polymerize G-actin (Fig. 1a, b and c). Histatin-3 fully polymerized 4 μM G-actin at 12 μM concentration (Fig. 1a and c) but more than 28 μM histatin-5 was needed to achieve a similar degree of polymerization (Fig. 1b). By high speed centrifugation 96 and 20% of F-actin incubated with 9 μM histatin-3 or histatin-5 was sedimented, respectively (Fig. 1c), showing again that histatin-3 is a better polymerizing agent than histatin-5. This is in spite of the fact that both histatins have the same number (5) of net positive charges and that the sequence of histatin-5 is identical with that of histatin-3 in the 1–24 N-terminal amino acids.
The histatin peptides are rich in histidine; both histatin-3 and histatin-5 contain 7 histidine residues. The pK of histidine is around pH 6.5 depending on the environment of the residue in the protein structure. This means that its charge changes with pH in the neutral pH region. Histidine is positively charged at slightly acidic pH and neutral above pH 7.0. Since actin polymerization is dependent on the number of positive charges of the polymerizing agent, the effect of pH 6.5, 7.4 and 8.2 on the polymerization of G-actin by histatins was measured (Fig. 1d). We found that the ability of both histatin-3 and histatin-5 to polymerize actin increased steeply with the decreasing pH. The pH effect on actin polymerization by histatins was compared to its effect on actin polymerization by LL-37, which does not contain histidine residues and at neutral pH polymerizes G-actin at about the same concentration as histatin-3 [9]. The kinetics of actin polymerization by LL-37 was found to be much less pH-dependent than the polymerization by histatins (Fig. 1d).
Bundling of actin filaments by histatin-3 and histatin-5
Polycations bundle F-actin filaments because their positive charge eliminates the repulsion between the negatively charged actin filaments [13]. We studied the kinetics of F-actin bundling by histatin-3 and −5 by light scattering (Fig. 2a). Histatin-3 and histatin-5 was found to bundle F-actin rapidly at stoichiometric (Fig. 2a), and superstoichiometric concentration (Fig. 2b), respectively. The dependence of the concentration of histatin-3 and −5 on the extent of actin filament bundling was measured by low speed centrifugation (Fig. 2c). We found that histatin-3 is the better bundling agent, since 14 μM of histatin-5, but only 5 μM of histatin-3 were required for 50% bundling 8 μM F-actin.
Bundling of actin filaments, like polymerization, depends on the number of net positive charges in the polycation, which is pH dependent in the histidine rich histatins. The bundling efficiency of histatins, as that of polymerization, was found to increase with the decreasing pH (Fig. 2d). This is contrary to the bundling by LL-37, whose sequence lacks histidine residues. The histatin concentration dependence on the extent of actin filament bundling was measured at pH 6.5 and 7.4 (Additional File 1: Figure S1). At pH 6.5 the bundling takes place at much lower concentrations of histatins than at pH 7.4. The effect of pH on the bundling by histatin-5 is much stronger than by histatin-3 (Fig. 2d and Additional File 1: Figure S1) in spite that both peptides contain the same number of histidine residues and net positive charges.
NaCl, DNase1 and cofilin unbundle (dissociate) histatin bundled F-actin
Polycation induced actin bundles are sensitive to ionic strength. They unbundle (dissociate) at increased salt concentration [8] because the salt masks the electrostatic interactions between the polycations and the negatively charged actin filaments [13]. The salt sensitivity of actin bundles is a measure of the relative role of electrostatic and hydrophobic interactions in the binding of polycation to actin. Low ionic strength sensitivity indicates hydrophobic binding between the cationic peptides and actin. Using low speed centrifugation we found that histatin-bundled actin filaments are easily unbundled at relatively low NaCl concentration (Fig. 3a). Bundles induced by 16 μM histatin-5 and histatin-3 are largely dissociated at 25 and 100 mM NaCl concentration, respectively (Fig. 3a). These results indicate that the actin-histatin interactions are basically electrostatic, but, hydrophobic interactions also have minor role in the binding of histatin-3 to actin. The kinetics of bundle dissociation was followed by light scattering, which showed that 200 mM NaCl completely dissociates actin filaments bundled by 8 μM histatin-3 (Fig. 3b) or 16 μM histatin-5 (Fig. 3c) in a few seconds.
DNase1 and cofilin disassemble LL-37 and lysozyme -induced actin bundles [9]. Both DNase1 and cofilin are actin binding proteins. DNase1 depolymerizes F-actin filament by binding to the protomer at the filaments ends and forming a tight complex with the protomer by attachment to its DNase1 binding loop (D-loop). The actin protomer (G-actin)-DNase1 complex gradually dissociates from the end of actin filament as shown by the disassembly of LL-37 induced actin bundles [9]. Cofilin disassembles actin bundles by severing the filaments through its effect on the structure of actin protomers [19, 20]. Light scattering results indicated that 4 μM F-actin bundled by 8 μM histatin-3 (Fig. 3b) or 16 μM histatin-5 (Fig. 3c) dissociates upon addition of 9 μM DNase1 or 9 μM cofilin. The speed of bundle dissociation was fastest with 200 mM NaCl, slower with 9 μM cofilin and slowest with 9 μM DNase (Fig. 3b and c). The light scattering decreased by 60% and 100% after 10 min of addition of 9 μM cofilin to actin filaments bundled by histatin-3 (Fig. 3b) and histatin-5 (Fig. 3c), respectively.
Effects of histatin-3 and histatin-5 on the fluorescence emission spectrum of TRC and DED labelled F-actin indicate that histatins affect F-actin structure (Fig. 4)
TRC and DED fluorescence labels bind covalently to Gln-41 in the D-loop of actin in a reaction catalyzed by transglutaminase [20–22]. The fluorescence emission spectra of these labels are sensitive to dynamic changes in actin structure. It was shown that cofilin significantly decreases the fluorescence emission intensity of TRC-labeled [22] and increases the intensity of DED-labeled F-actin [20] due to its effect on the dynamic structure of the D-loop in the subdomain-2 of actin. We found that also histatins affect the fluorescence emission of TRC-F-actin (Fig. 4a and b) and compared these effects with those induced by cofilin. Cofilin-induced fluorescence decrease was found to be significantly greater than that caused by histatins. The effect of histatins and cofilin on the spectrum is additive. Histatin-3 has a larger effect on TRC-F-actin fluorescence than histatin-5, as 8 μM histatin-3 decreases the fluorescence emission at 581 nm (fluorescence emission maximum) by 22.3%, while 16 μM histatin-5 by 18.1%. The effect of histatins and cofilin on the dynamic structure of F-actin was also studied by recording the changes in the fluorescence emission spectrum of DED-label attached to Gln-41 of actin [21]. We found that both histatins and cofilin increase the intensity and blue shift the fluorescence emission spectrum of DED-F-actin (Fig. 4c and d). Both the intensity increase and the blue shift caused by cofilin are significantly greater than those induced by histatins. Histatins move the peak of the spectrum from 528 to 519 nm, while cofilin moves it to 501 nm. The effect of cofilin and histatins on the DED-F-actin spectrum is additive as addition of histatins to cofilin treated F-actin cause further fluorescence intensity increase (Fig. 4c and d). However, the addition of histatin-5 induces much greater increase in the intensity cofilin treated DED-F-actin than that of histatin-3. The histatins and cofilin induced changes in the fluorescence emission spectra of TRC- and DED-F-actin point to alterations in the D-loop structure of actin and in the dynamic structure of F-actin in general. However, there are also differences in histatins and cofilin caused spectral changes, since cofilin induces much larger change in fluorescence intensity in both TRC- and DED-F-actin and significantly greater blue shift in the spectrum of DED-F-actin than histatins do. This indicates that cofilin induces more significant alterations in the dynamic structure of F-actin than histatins. The results also show that the histatin-3 and −5 induced structural changes are not identical as histatin-3 cause larger change in the spectrum of TRC-F-actin than histatin-5, while histatin-5 increases more the emission fluorescence of DED-F-actin in the presence of cofilin than that of histatin-3.
Histatin-3 and histatin-5 are cross-linked to both G- and F-actin by transglutaminase
Transglutaminase (TGase) catalyzes the formation of isopeptide bonds, which cross-links proximal glutamine and lysine residues in proteins. The TGase reaction produces both intramolecular cross-links in proteins and intermolecular cross-links between proteins and peptides [23]. Transglutaminase treatment of actin has been used to study the structure and function of both monomeric (G) and polymeric (F) actin [21, 24]. We found earlier that LL-37 can be cross-linked to actin by TGase [6, 25]. Here we compared the cross-link formation of histatin-3 and LL-37 with G- and F-actin (Fig. 5a). Histatin-3 was found to be cross-linked to both G- and F-actin like LL-37, but in all cases the extent of cross-link formation with F-actin was significantly less than with G-actin. The bands of a single histatin-3 molecule cross-linked to actin (cross-linked product molecular weight 45 K) and two molecules of histatin-3 cross-linked to actin (48 K molecular weight) were visualized on SDS-PAGE. These products are similar to those obtained by LL-37. However, the bands of actin dimer-peptide and actin higher oligomers-peptide cross-link were missing or weak with histatin-3. We studied the cross-link formation of G-actin also with histatin-5. In this case the actin-single peptide cross-linked band is not well separated from the band of actin because of the low molecular weight of histatin-5 (Fig. 5b). Therefore we used the fluorescence (Fl) derivatives of the histatins for the quantitative study of the cross-linking. With the fluorescence derivatives, the histatin-actin cross-linked bands could be seen without the actin band in fluorescent SDS-PAGE. In Coomassie blue stained SDS-PAGE the separation of Fl-histatin-5-actin monomer cross-linked band from the actin band is better than that of the non-fluorescent histatin-5 because the added mass of the fluorescence moiety (Fig. 5b). The extent of cross-linking is slightly larger with the fluorescent than with the non-fluorescent histatins. Comparing the cross-linking of Fl-histatin-3 and Fl-histatin-5 with G-actin we noticed that the quantity of the Fl-histatin-5-actin cross-linked product is consistently greater than that of Fl-histatin-3 (Fig. 5b). To compare histatin-actin cross-links with LL-37-actin cross-link histatin-3 and −5 were competed with Fl-LL-37 for cross-linking to G-actin (Fig. 5c). We found that 3 μM histatin-3 or histatin-5 decreased the extent of cross-linking of 4 μM G-actin with 6 μM Fl-LL-37 by 92 and 68% percent, respectively. The findings that at substoichiometric concentrations of histatins relative to Fl-LL-37, inhibited more than 50% of the cross-linking of Fl-LL-37, indicate that the ability of histatins to cross-link G-actin is greater than that of LL-37. Since LL-37 binds preferentially to the D-loop of actin [6], the competition of histatins with LL-37 for actin cross-linking may suggest that histatin-3 and −5 also bind to the same actin loop.
Kinetics of cross-link formation and the effect of phalloidin on the extent of cross-link between actin and Fl-histatins
The kinetics (Fig. 6a) and extent (Fig. 6b) of the TGase catalyzed cross-link formation of Fl-histatins to G- and F-actin was studied. The extent of cross-linking of Fl-histatin-5 to both G- and F-actin was larger than that of Fl-histatin-3 (Figs. 5b and 6). The cross-linking of Fl-histatins to G-actin was faster and its extent significantly larger than to F-actin (Fig. 6). As the result of treadmiling [26] there is always a small concentration of G-actin in F-actin preparation and equilibrium exist between the two forms. Because of the large difference in the extent of cross-linking of histatins to G- and F-actin one may assume that also in the case of F-actin, the G-actin, which is present in the F-actin preparation, reacts with histatins. The actual cross-linking of histatins to F-actin might be rather minor. To check this assumption we cross-linked F-actin with histatins and LL-37 also in the presence of phalloidin (Fig. 6b). Phalloidin inhibits treadmiling and decreases the concentration of G-actin present in the F-actin preparations [27]. We found that phalloidin reduces greatly the extent of F-actin cross-linking with Fl-histatin-3, Fl-histatin-5 and Fl-LL-37 (Fig. 6b), which supports the above hypothesis. The poor cross-linking of histatins and LL-37 to F-actin is caused by the structural changes taking place in the D-loop of actin during polymerization.
NaCl, DNase1 and cofilin inhibit the cross-linking of histatins to G-actin
We found that NaCl, DNase1 and cofilin unbundles histatin-3, histatin-5 (Fig. 3) and LL-37 [9] induced F-actin bundles. Here we studied the effect of these agents on the cross-link formation between G-actin and histatins. In these experiments NaCl, histatins, TGase were added simultaneously to G-actin and incubated for 1 min only to avoid significant actin polymerization. NaCl inhibited the cross-linking of Fl-histatin-3 and −5 to G-actin (Fig. 7a), but its effect was less than that on dissociation of bundles (Fig. 3). Addition of 200 mM NaCl decreased the extent of cross-linking by histatins more than 70% (Fig. 7a) but did not affect the extent of cross-linking by LL-37 [6]. These results support the conclusions of the unbundling experiments with NaCl (Fig. 3a) that the binding of histatins to actin is less hydrophobic than that of LL-37.
DNase1, which binds to D-loop of actin, strongly inhibits cross-link formation between G-actin and histatins (Fig. 7b). The strong inhibitory effect of DNase1 on the histatin-actin cross-linking indicates (see also Fig. 5b) that histatin 3 and −5 bind to the DNase1 binding loop (D-loop) of G-actin. Cofilin, which partially binds to the DNase binding loop actin [28], only slightly inhibits the Fl-histatin-G-actin cross-linking (Fig. 7b). The inhibitory effect of cofilin was less Fl-histatin-5 than with Fl-histatin-3.
Histatin cross-linking sites on G-actin
TGase catalyzes covalent cross-linking of glutamine and lysine residues in proteins [23]. Since in the histatin-G-actin cross-linking reaction only actin contains glutamine residues, it follows that actin should be the glutamine donor in this case. DNase1, which binds to the D loop (His40-Lys50 sequence) of G-actin [29], strongly inhibits cross-linking of histatins to actin (Fig. 7b). These results indicate that histatin-3 and −5 also bind to the D-loop of G-actin and should be cross-linked to either Gln41 or Gln49 in the D-loop. Gln41 seems to be the preferred candidate for this role since this residue participates in the intrastrand cross-linking by N-(4-azido-2-nitrophenyl) putrescine between F-actin protomers [30] and in the intramolecular actin cross-linking to Lys50 [24]. This residue was also labeled with DED [21] and TRC [22] fluorescent probes by TGase. On the other hand LL-37, which also binds to the D-loop [6], is preferentially cross-linked to Gln-49 [6]. The cross-linking of Fl-LL-37 to G-actin is inhibited by histatin-3 and −5 (Fig. 5c). To locate the cross-linking site in the D-loop we cross-linked G-actin and TGase pretreated G-actin (TG-G-actin) with Fl-histatin-3 and −5 (Fig. 8). The TGase treatment of G-actin yields intramolecular cross-link between Gln41 and Lys50 and makes Gln41 unavailable for further cross-linking [24]. We found that slightly less actin Fl-histatin-3 cross-linked products formed with TGase pretreated than with untreated G-actin. (Fig. 8). Therefore, the result that TGase pretreatment only slightly decreases the cross-link formation between G-actin and Fl-histatin-3 indicates that Gln-49 is the main glutamine donor in the D-loop of actin in this reaction. However, a minor role of Gln41 cannot be excluded in this cross-linking. On the other hand, the same amount of cross-linked product formed in the reaction of G-actin or TG-G-actin with Fl-histatin-5 (Fig. 8) indicating that Fl-histatin-5 does not cross-link to Gln41 of G-actin.
Subtilisin cleaves G-actin into Asp1-Met47 N-terminal and Gly48-Phe375 C-terminal fragments [31]. We found that both fragments can be cross-linked with Fl-histatin-3 (Fig. 9a). The N-terminal fragment contains Gln41 as single glutamine residue which proves that Gln-41 can also be glutamine donor in cross-linking of Fl-histatin-3 to G-actin. However, Fl-histatin-5 is cross-linked only to the C- but not to the N-terminal G-actin fragment (Fig. 9b), which supports our finding that Fl-histatin-5 is not cross-linked to Gln41 of G-actin (Fig. 8). The cross-link formation between Fl-histatin-3 or Fl-histatin-5 and the C-terminal actin fragment (Fig. 9), which contains the other D-loop glutamine, Gln49, indicates that this residue is the main cross-linking site on the D-loop of G-actin. Trypsin cleaves G-actin between Lys68 and Tyr69 leading to the formation of stable Tyr69-Arg372 C-terminal fragment [31]. We found that the Tyr69-Arg372 C-terminal tryptic fragment, which does not contain the D-loop, can be also cross-linked to both Fl-histatin-3 and −5 (Fig. 9). This finding indicates that beside Gln41 and Gln49 other, non-D-loop glutamines of actin, can also serve as minor glutamine donors in the TGase catalyzed actin-histatin cross-linking reaction.