Two Different Heparin-binding Domains in the Triple-helical Domain of ColQ, the Collagen Tail Subunit of Synaptic Acetylcholinesterase
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m301384200
ISSN1083-351X
AutoresPaola Deprez, Nibaldo C. Inestrosa, Éric Krejci,
Tópico(s)Enzyme function and inhibition
ResumoColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina. ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina. At vertebrate cholinergic synapses, acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; HBD, heparin-binding domain; HS, heparan sulfate; HSPG, HS proteoglycan; WT, wild-type. rapidly hydrolyzes the neurotransmitter acetylcholine, thereby terminating synaptic transmission. This key function does not only require a high catalytic turnover number but also a strategic positioning of the enzyme. This is achieved by the association of AChE catalytic subunits with structural subunits that bring them to the site of action. In the case of asymmetric AChE, the collagen ColQ is responsible for the localization of the enzyme at the vertebrate neuromuscular junction. Inactivation of the ColQ gene in mice or mutations in the human ColQ gene result in the absence of enzyme accumulation at the neuromuscular junction and are the cause of a congenital myasthenic syndrome (type 1c) (1Feng G. Krejci E. Molgo J. Cunningham J.M. Massoulié J. Sanes J.R. J. Cell Biol. 1999; 144: 1349-1360Google Scholar, 2Ohno K. Engel A.G. Brengman J.M. Shen X.-M. Heidenreich F. Vincent A. Milone M. Tan E. Demirci M. Walsh P. Nakano S. Akiguchi I. Ann. Neurol. 2000; 47: 162-170Google Scholar). As found in other collagens, ColQ contains a central triple-helical domain surrounded by non-collagenous N- and C-terminal domains (Fig. 1A). Each N-terminal domain organizes an AChE tetramer, so the triple-helical structure generates an A12 or asymmetric AChE form with 12 catalytic subunits (3Krejci E. Coussen F. Duval N. Chatel J.M. Legay C. Puype M. Vandekerckhove J. Cartaud J. Bon S. Massoulié J. EMBO J. 1991; 10: 1285-1293Google Scholar, 4Bon S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Google Scholar). The collagen domain is characterized by Gly-Xaa-Yaa repeats and a high proportion of the stabilizing proline and hydroxyproline residues. The C-terminal domain is divided into a proline-rich region, important for triple-helix formation (5Bon S. Ayon A. Leroy J. Massoulié J. Neurochem. Res. 2003; 28: 523-535Google Scholar), and a cysteine-rich region probably involved in the anchorage of AChE at the neuromuscular junction, since mutations in this region prevent the accumulation of AChE in congenital myasthenic syndrome type 1c patients (2Ohno K. Engel A.G. Brengman J.M. Shen X.-M. Heidenreich F. Vincent A. Milone M. Tan E. Demirci M. Walsh P. Nakano S. Akiguchi I. Ann. Neurol. 2000; 47: 162-170Google Scholar). Heparan sulfate proteoglycans (HSPGs) have been implicated in the anchorage of asymmetric AChE to the synaptic basal lamina by interacting with ColQ through their heparan sulfate (HS) chains. This was proposed after showing that AChE could be specifically solubilized from the tissue with heparinase as well as with HS and heparin (6Brandan E. Maldonado M. Garrido J. Inestrosa N.C. J. Cell Biol. 1985; 101: 985-992Google Scholar). Consistently, binding of the enzyme to the cell surface is inhibited after treatment of the myotubes with heparitinase and in cells deficient in HS synthesis (7Inestrosa N.C. Gordon H. Esko J.D. Hall Z.H. Shafferman A. Velan B. Multidisciplinary Approaches to Cholinestease Function. Plenum Press, New York1992: 25-32Google Scholar). Later studies showed that exogenously added asymmetric AChE associated specifically with nerve-muscle contact sites in a heparin-sensitive manner (8Rotundo R.L. Rossi S.G. Anglister L. J. Cell Biol. 1997; 136: 367-374Google Scholar), suggesting that HSPGs would define its localization at the neuromuscular junction. Perlecan, a basal lamina HSPG, has been proposed as the receptor of collagen-tailed AChE (9Peng H.B. Xie H. Rossi S.G. Rotundo R.L. J. Cell Biol. 1999; 145: 911-921Google Scholar), consistent with the recent finding that AChE is not accumulated at the neuromuscular junction in perlecan-null mice (10Arikawa-Hirasawa E. Rossi S.G. Rotundo R.L. Yamada Y. Nat. Neurosci. 2002; 5: 119-123Google Scholar). In this context, we have found that the collagen domain of ColQ contains two heparin-binding consensus sequences of the BBXB form (where B represents a basic residue) surrounded by other basic residues and proposed they constitute the sites for HSPG recognition (11Deprez P. Inestrosa N.C. J. Biol. Chem. 1995; 270: 11043-11046Google Scholar). We have characterized the structural properties of those putative heparin-binding domains (HBDs) by using molecular modeling and synthetic peptides (12Deprez P. Inestrosa N.C. Protein Engng. 2000; 13: 27-34Google Scholar, 13Deprez P. Doss-Pepe E. Brodsky B. Inestrosa N.C. Biochem. J. 2000; 350: 283-290Google Scholar, 14Doss-Pepe E. Deprez P. Inestrosa N.C. Brodsky B. Biochemistry. 2000; 39: 14884-14892Google Scholar). Here, we analyzed ColQ-heparin interactions by heparin affinity chromatography of the entire A12 AChE, carrying different mutations in ColQ. We first established that recombinant and purified A12 enzymes share the same properties. Then we dissected the participation of the different domains of ColQ as well as of the individual basic residues composing both HBDs to define the determinants of heparin binding. The results obtained provide evidence that expands the role of ColQ-HSPGs interactions to the fine ultrastructural organization of AChE at the synaptic basal lamina. Moreover, they contribute to our understanding of the nature of a HBD in the context of the canonical structure of collagen. Materials—All restriction endonucleases were purchased from New England Biolabs (Ozyme, France). Other molecular biology reagents were from Promega Corp. (Madison, WI). Oligonucleotides were synthesized either by Oligo Express or Eurobio (Paris, France). Heparin-agarose was purchased from Pierce. Heparin-albumin was obtained from Sigma. Acridine-agarose was kindly provided by Dr. Terry Rosenberry (Mayo Clinic, Jacksonville, FL). Unless otherwise specified, other reagents were obtained from commercial sources. Site-directed Mutagenesis—Two different plasmids were constructed using the coding sequences for rat AChE and ColQ1a (15Legay C. Bon S. Vernier P. Coussen F. Massoulié J. J. Neurochem. 1993; 60: 337-346Google Scholar, 16Krejci E. Legay C. Thomine S. Sketelj J. Massoulié J. J. Neurosci. 1999; 19: 10672-10679Google Scholar). Both coding sequences were inserted between 5′- and 3′-untranslated sequences of Xenopus globin mRNA and then subcloned into the pcDNA3 vector (Invitrogen). The recombinant vector pcDNA3-ColQ1a served as a template for site-directed mutagenesis, performed as described by Kunkel (17Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Google Scholar). To confirm the presence of desired mutations, all mutant plasmids were sequenced by Génome Express (Montreuil sous Bois, France). Expression in Xenopus Oocytes—Capped synthetic transcripts were prepared using the Ambion mMESSAGE mMACHINE™ in vitro transcription kit (Austin, TX). Samples of ∼50 nl containing 2.5 ng of AChE mRNA and different amounts of ColQ mRNA were injected with a Drummond Nanojet injection system (Broomall, PA) into the animal poles of Xenopus oocytes (18Soreq H. Parvari R. Silman I. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 830-834Google Scholar). Injected oocytes were incubated at 18 °C for 24–48 h in Barth medium (5 mm HEPES, pH 7.6, 98 mm NaCl, 1.8 mm CaCl2, 1 mm MgCl2, and 50 μg/ml gentamycin) supplemented with 50 μg/ml ascorbic acid to promote triple-helix formation. Extraction and Isolation of Recombinant AChE—Injected Xenopus oocytes were homogenized by repeated pipetting in 10 μl/oocyte of ice-cold extraction buffer containing 50 mm Tris-HCl, pH 7.0, 10 mm MgCl2, 0.8 m NaCl, 1% Brij-96. Different AChE molecular forms were isolated by velocity sedimentation in 5–20% linear sucrose gradients containing extraction buffer. Sucrose gradients were centrifuged at 40,000 rpm for 15 h at 7 °C in a SW41 Beckman rotor (Fullerton, CA) and were collected in ∼48 fractions of ∼320 μl each from which 10 μl/fraction were used to measure AChE activity (19Ellman G.L. Courtney K.D. Andres V.J. Featherstones R.M. Biochem. Pharmacol. 1961; 7: 88-95Google Scholar). The sedimentation coefficients for AChE forms were determined from those of β-galactosidase (16 S) and alkaline phosphatase (6.1 S) from Escherichia coli, run in the gradients as internal markers. Fractions containing the different AChE forms were pooled and stored at –80 °C for future analysis. For comparison, the different forms of AChE isolated from the sucrose gradient were further purified by affinity chromatography using an acridine-agarose column as for enzyme extracted from Torpedo electric organ, but since they showed exactly the same behavior as the enzyme isolated directly from the sucrose gradients, this enzyme was more often used for the experiments. Purification of Torpedo Asymmetric AChE—Asymmetric AChE was purified from Torpedo californica electric organ (Pacific Bio-Marine Laboratory, Venice, CA) using sequential extraction and affinity chromatography, as described previously (13Deprez P. Doss-Pepe E. Brodsky B. Inestrosa N.C. Biochem. J. 2000; 350: 283-290Google Scholar). Heparin-Agarose Affinity Columns—Relative affinity for heparin was analyzed for different ColQ mutants using heparin-agarose affinity columns, where the bound enzyme was eluted with a linear NaCl gradient. After packing 500 μl of heparin-agarose in a column without flow, the affinity resin was equilibrated with 50 mm Tris-HCl, pH 7.4, and 100 mm NaCl, and 20–60 milliunits of asymmetric AChE in equilibration buffer were loaded. The column was washed with 10 ml of equilibration buffer, and the enzyme was finally eluted using a 20-ml linear NaCl gradient of either 0.1–0.8 m NaCl or 0.2–0.9 m NaCl in 50 mm Tris-HCl, pH 7.4. Loading, washing, and elution steps were performed at a constant flow rate of 9.75 ml/h, maintained by a Gilson Miniplus 2 peristaltic pump. Approximately 77 fractions of ∼260 μl each were collected. For all of these, 100-μl aliquots were used to determine AChE activity, whereas aliquots of 100 μl diluted with 1 ml of distilled water were used to measure conductivity using an Amber Science Inc. 605 conductivity meter (Eugene, OR). Conductivity values were converted to NaCl concentrations using a 5-point standard curve with NaCl solutions made up with elution buffer. NaCl concentrations increased by 0.0105 ± 0.0006 m/fraction (n = 97). Equilibrium Binding Assays—Binding assays were performed in MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) coated with heparin coupled to albumin as described previously (13Deprez P. Doss-Pepe E. Brodsky B. Inestrosa N.C. Biochem. J. 2000; 350: 283-290Google Scholar). After coating and blocking steps, the plates were incubated with 0.25 milliunits of recombinant A12 AChE (non-saturating amount of enzyme) in 50 μl of 50 mm Tris-HCl, pH 7.4, and 150 mm NaCl for 18 h at 4 °C with constant agitation. The supernatant was recovered, the plates were washed twice with 100 μl of incubation buffer for 5 min, and AChE activity was measured in the supernatant and washes (both representing the unbound enzyme) as well directly in the plates (bound enzyme). Data Treatment—To analyze the peak composition of elution profiles of AChE from heparin-agarose columns, the noise present in raw data was filtered using the Savitzky-Golay algorithm, the base line was detected and subtracted using a non-parametric algorithm, and finally, the peaks were detected and fitted to Gaussian peaks using the method of the second derivative (r2 > 0.999) using the program PeakFit™ 4.06 from SPSS Inc. (Chicago, IL). To facilitate the analysis of elution profiles of AChE from heparin-agarose and the comparison between different asymmetric AChE mutants, raw data from AChE elution profiles were plotted for AChE activity versus NaCl concentration, fitted to a non-parametric base line that was then subtracted, and converted to cumulative areas using the program PeakFit™ 4.06 from SPSS Inc. (Chicago, IL). Using the Origin® 6.0 program from Microcal Software Inc. (Northampton, MA), cumulative area values were normalized to percentages and the data plotted as cumulative areas (%) versus logarithm of NaCl concentration (m) were fitted to a dose-response (or four parameter) sigmoidal equation with variable slope, y=A1+A2−A11+10(logx0−x)p(Eq. 1) where the parameters A1, A2, log x0, and p correspond to the bottom asymptote, the top asymptote, the center, and the variable Hill slope, respectively. Finally, starting from the center parameter value (i.e. log[NaCl]), the NaCl concentration for 50% elution of the bound enzyme was obtained for each curve. This value was used in this study as a measure of the relative affinity of a given A12 AChE mutant for heparin. In the different experiments, statistical analyses were performed using the unpaired Student's t test. Production of Recombinant Rat A12AChE and Its Affinity for Heparin—Xenopus oocytes constitute a very efficient system to produce predominantly A12 AChE forms by injecting defined amounts of both ColQ and AChE mRNAs (16Krejci E. Legay C. Thomine S. Sketelj J. Massoulié J. J. Neurosci. 1999; 19: 10672-10679Google Scholar), as shown by the sedimentation profile in a sucrose gradient of oocyte extracts (Fig. 2A). To evaluate the heparin-binding behavior of recombinant rat asymmetric AChE, its A12 form produced in Xenopus oocytes was isolated from a sucrose gradient, bound to a heparin-agarose column, and eluted from the resin using a linear NaCl gradient. This strategy allows determination of the values of relative affinity for heparin, where a protein with higher affinity is eluted at a higher NaCl concentration (20Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Google Scholar). A12 AChE purified from Torpedo electric organ was also analyzed as reference. As shown in Fig. 2, B and C, both Torpedo purified and rat recombinant A12 AChE present the same elution profile, composed of five peaks eluting at the same NaCl concentrations and presenting similar relative abundance. In both cases the majority of the enzyme was eluted in the central peak at a concentration of 0.6 m NaCl. The same elution profile was obtained under different experimental conditions, varying the NaCl concentration increment per fraction, the elution volume, the elution flow rate, and the volume of resin (data not shown). Each peak differed significantly from its neighboring peaks both at the level of NaCl concentration of elution and relative abundance (n = 12, p < 0.0003). This multiplicity might reflect an intrinsic heterogeneity of enzyme species. To examine this possibility, AChE eluted in the first peak was re-loaded onto a second heparin-agarose column, generating the original five peaks in the elution profile (data not shown). This excludes the possibility that the enzyme is composed of chemically different species. Most likely, the observed behavior arises from the multivalent nature of the HBDs in ColQ (12Deprez P. Inestrosa N.C. Protein Engng. 2000; 13: 27-34Google Scholar) and the heterogeneity of heparin molecules. To facilitate the analysis of the elution profiles of AChE from heparin-agarose and the comparison between different A12 AChE mutants, the elution profiles were expressed as cumulative areas versus log[NaCl]. The NaCl concentrations at which 50% of the bound enzyme was eluted were derived from these curves (see "Experimental Procedures"), and relate directly to their affinity for heparin. When expressed in this form, both Torpedo and recombinant rat enzymes presented the same elution curve, with 50% of the enzyme eluting at 0.6 m NaCl (Fig. 2D, Table I). These results show that the recombinant rat A12 AChE behaves exactly as the purified A12 enzyme from Torpedo electric organ, so that Xenopus oocytes constitute a good expression system for A12 AChE.Table INames, description, and relative affinity of the different A12 AChE variants Open table in a new tab The Heparin-binding Activity Is Localized in the Collagen Domain of ColQ—By analyzing the ColQ sequence, it is possible to identify two heparin-binding consensus sequences of the BBXB type in the collagen domain of ColQ. Other clusters of basic residues also exist within the N- and C-terminal non-collagenous domains of ColQ, and it has been suggested that the C-terminal domain could participate in binding to HSPGs (2Ohno K. Engel A.G. Brengman J.M. Shen X.-M. Heidenreich F. Vincent A. Milone M. Tan E. Demirci M. Walsh P. Nakano S. Akiguchi I. Ann. Neurol. 2000; 47: 162-170Google Scholar). To define the contribution of different domains to heparin binding, AChE oligomers containing ColQ with different deletions were expressed and analyzed. In ΔCt, the last 88 residues of ColQ were deleted, corresponding to the cysteine-rich region of the C-terminal domain (Fig. 1B). The remainder of the C-terminal domain was not removed because it is required for triple-helix folding. ΔCol contains a deletion of 171 amino acids in the central region of the collagen domain of ColQ that eliminates the two HBDs, but 7 proline-rich Gly-Xaa-Yaa triplets were left to ensure the formation of a stable triple helix. Finally, QN contained only the N-terminal domain of ColQ, allowing the organization of AChE tetramers to occur independently of the collagen domain. Fig. 3A shows the sedimentation profiles of the different ColQ deletion mutants co-expressed with AChE in Xenopus oocytes. As expected, ΔCt and ΔCol were able to produce asymmetric forms of AChE, whereas QN only produced a tetramer of catalytic subunits (G4 form). AChE expressed with ΔCt presented a sedimentation profile similar to wild type (WT), whereas asymmetric forms produced with ΔCol presented higher sedimentation coefficients, consistent with a decreased asymmetry of the molecules due to a shortened collagen tail. The A12 ΔCol and ΔCt forms as well as the QN G4 form were isolated from the sucrose gradients and run on heparin-agarose columns. As shown in Fig. 3B, QN and ΔCol completely lost their ability to bind heparin, whereas ΔCt presented an elution profile similar to WT (Fig. 3C). The NaCl concentrations at which 50% of the total bound enzyme eluted was 0.61 m for the WT enzyme and 0.63 m for ΔCt, not differing significantly (see Table I for mutant sequences, nomenclature, and elution values and Fig. 9 for a summary of relative heparin affinity of all the mutants). Together, these results clearly show that the heparin-binding activity of asymmetric AChE resides exclusively in the collagen domain of ColQ. The Basic Residues of Both HBDs in the Collagen Domain of ColQ Interact with Heparin—To confirm that the heparin-binding consensus sequences present in the collagen domain of ColQ are responsible for interactions with heparin, double site-directed mutagenesis was carried out. The first two basic residues of both BBXB consensus sequences were replaced by aspartic acid and proline (N–/C–, Table I). Aspartic acid was chosen to obtain a dramatic effect because the opposite charge was likely to cause heparin repulsion. Proline was used as it lacks a reactive side chain and tends to play a structural rather than a functional role (21Shah N.K. Ramshaw J.A. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Google Scholar). Only 55% of N–/C– AChE was retained in heparin-agarose, with the bound enzyme eluting at 0.17 m NaCl (Fig. 4, A and B). This suggests that the BBXB sequences in ColQ may be essential for heparin interactions under physiological conditions. Indeed, an equilibrium binding assay performed at 0.15 m NaCl using heparin-coated plates showed that only 16% of the N–/C– enzyme was able to bind heparin (Fig. 4A). On the other hand, the fact that N–/C– still presented some heparin-binding capacity suggested that the basic residues surrounding the BBXB motif may also participate in this interaction. To assess this possibility, almost all basic residues constituting both putative HBDs were substituted by alanines (N=/C=, Table I). In this case, only 14% of the N=/C= mutant enzyme was retained by heparin-agarose, with only 5% able to bind heparin under equilibrium conditions (Fig. 4A). These results demonstrate that the N- and C-terminal HBDs, comprising BBXB motifs surrounded by other basic residues and localized in the collagen domain of ColQ, are responsible for the interaction of A12 AChE with heparin. The Two HBDs in the Collagen Domain of ColQ Have Different Affinities for Heparin—To analyze the behavior of each HBD separately, two ColQ mutants were generated by interrupting a single BBXB motif and leaving the other intact. Thus, N–/C+ allowed the analysis of the C-terminal HBD and N+/C– of the N-terminal HBD (Fig. 1B). In both cases, the modified BBXB sequence was substituted by DPXB (Table I). Both mutants were fully retained in heparin-agarose as the WT enzyme but eluted from the column at different NaCl concentrations (Fig. 5A). Whereas the N–/C+ enzyme eluted at 0.54 m NaCl, the value for N+/C– was 0.41 m, implying that the C-terminal HBD has a significantly higher affinity for heparin than the N-terminal HBD. Interestingly, both N–/C+ and N+/C– mutants presented lower affinities for heparin than the WT enzyme (0.61 m), suggesting that WT affinity results from the simultaneous or cooperative participation of both HBDs. The Difference between the Two HBDs Does Not Simply Result from the Different Number or Nature of Basic Residues— Although ColQ HBDs share a minimum GRPBBXB sequence, the two sites differ both in the number and nature of basic residues. The C-terminal HBD presents two additional basic residues that are not found in the N-terminal HBD. To evaluate whether these extra basic residues could explain the higher heparin affinity of the C-terminal HBD, these residues were replaced by alanines (N–/R218A-K233A, Table I). Fig. 5B shows that the resultant mutant had a slightly lower affinity for heparin than N–/C+, eluting at a concentration of 0.51 m NaCl. This suggests that the extra basic residues at the C-terminal HBD participate in heparin interactions even if their contribution is small. However, N–/R218A-K233A presented a heparin affinity significantly higher than that of the N-terminal HBD (N+/C–), showing that the basic charge difference between the two HBDs does not underlie their different affinities for heparin. The second difference between the two HBDs lies in the identity of basic residues that constitute each BBXB motif: RKGR for the N-terminal and KRGK for the C-terminal domain, with both sequences conserved among species. To assess whether this could explain the different affinities for heparin, we designed two new ColQ mutants in which the BBXB motifs were exchanged between the sites. Having eliminated the C-terminal motif, the remaining N-terminal BBXB sequence was replaced by that of the C terminus and vice versa (Fig. 1B, Table I). As shown in Fig. 5B, the C→N/C– mutant, which presents only the C-terminal BBXB motif located at the N-terminal position, eluted from the heparin-agarose column at the same NaCl concentration as N+/C–. Similarly, N–/N→ C, which presents the N-terminal consensus sequence at the C-terminal position, behaved exactly as did N–/C+. These results indicate that the different heparin affinities of the two HBDs are not explained by the identities of the basic residues that constitute each BBXB motif. Contribution of Individual Basic Residues to Heparin Interactions—To evaluate individual residue contributions within the two binding sites, point mutations were carried out in each HBD to eliminate basic charges while trying to alter the local conformation as little as possible. Arginine or lysine residues located in the Xaa position as well as lysines in the Yaa position of Gly-Xaa-Yaa triplets were substituted by alanines, whereas proline was used to replace arginines in the Yaa position. Previous studies show arginine and proline in the Yaa position to be equally stabilizing, whereas the stability of arginine in the Xaa position as well of lysines in both positions were equivalent to alanine (22Yang W. Chan V.C. Kirkpatrick A. Ramshaw J.A. Brodsky B. J. Biol. Chem. 1997; 272: 28837-28840Google Scholar). As shown in Fig. 6A, all N-terminal HBD mutants displayed decreased heparin affinities, suggesting the involvement of all residues from this HBD in heparin binding. According to the relative affinity loss presented by each mutant, the importance of each basic residue was ranked as Lys-122 > Arg-124 > Arg-118 ≥ Arg-121. In the C-terminal HBD, all mutations generated a significant change in heparin affinity (Fig. 6B). The mutation of any basic residue from the common GRPGBBGB sequence led to a decrease in heparin affinity. Residues were ranked in the following order of importance: Lys-226 > Lys-229» Arg-227 » Arg-223. Unexpectedly, the mutation of the most distal surrounding basic residues led to an increase in heparin affinity. The NaCl concentration required to elute N–/R218P did not differ significantly from the WT enzyme, in which both HBDs are intact, whereas that of N–/K233A surpassed it (Table I). Effect of Local Stability on Heparin Binding—From results shown in Fig. 5B, it is clear that neither the amount of basic charge nor the nature of the basic residues in the HBDs is the only determinant for heparin affinity, suggesting that structural factors such as local conformation could play a role. Proline is the most stabilizing residue found in collagens (21Shah N.K. Ramshaw J.A. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Google Scholar). Hence, basic residues from each HBD were selected to be replaced alternatively with alanine or proline. In the N-terminal HBD, substitution of the central lysine by proline caused a small affinity loss in contrast to the dramatic decrease observed for alanine substitution (Fig. 7A). In the C-terminal HBD, substitution of Arg-227 by proline induced a lower decrease in heparin affinity than substitution by alanine. For Arg-218, mutation to alanine decreased the affinity, whereas mutation to proline increased the affinity for heparin (Fig. 7B). In each case, affinity was higher with a proline than with an alanine. Contribution of the Distant Environment to Heparin Binding—Because local conformation seemed to be important in modulating heparin affinity, the possibility that regions distant from the HBDs could influence ColQ-heparin interactions was investigated. For this, ColQ deletions were designed to subject each HBD and its local environment to the same distant environment. The local environment was defined as the five triplets flanking each BBXB motif, so that each broader "mega"-HBD comprised 12 triplets. A single mega-HBD was then conserved, and the entire sequence (35 triplets) between the N-terminal limits of these domains was deleted (Figs. 1B and 8A). Thus, ΔN contained the C-terminal HBD and vice versa for ΔC, both surrounded by the most C-terminal and N-terminal regions of ColQ. Both constructs were able to produce truncated asymmetric AChE forms in Xenopus oocytes, exhibiting identical sedimentation profiles in sucrose gradients characterized by increased S values (Fig. 8B). As shown in Fig. 8C, ΔN eluted from the heparin column at 0.5 m NaCl, a value lower than that of N–/C+, whereas the ΔC mutant eluted at 0.47 m NaCl, higher than N+/C– (see Fig. 9 for a summary of relative heparin affinity of all the mutants). This indicates tha
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