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1 Dipartimento di Chimica, Università di Salerno, Salerno, Italy
2 Dipartimento di Pediatria, Università di Napoli "Federico II," Naples, Italy
Reprint requests to: Carla Esposito, Dipartimento di Chimica, Università di Salerno, Via S. Allende, 84081 Baronissi, Salerno, Italy; e-mail: cesposito{at}unisa.it; fax: 0039-089-965296.
(RECEIVED May 21, 2002; FINAL REVISION July 30, 2002; ACCEPTED September 24, 2002)
3 Present address: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II," Naples, Italy. 
Article and publication date are at www.proteinscience.org/cgi/doi/10.1110/ps.0216103.
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Keywords: Glyceraldehyde 3-phosphate dehydrogenase; lysine residues; mass spectrometry; Qn disease; transglutaminase
Abbreviations: Asp-N, endoproteinase Asp-N • CD, circular dichroism • ESIMS, electrospray mass spectrometry • GAPDH, glyceraldehyde 3-phosphate dehydrogenase • GEE, glycine ethylester • HD, Huntington’s disease • HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol • Q, glutamine residue • K, lysine residue • LC/MS, liquid chromatography mass spectrometry • MALDI/MS, matrix assisted laser desorption ionization mass spectrometry • MDC, monodansylcadaverine • Qn, polyglutamine repeat containing n Q residues • Q17, peptide with sequence RPRPRQ17RPRPR • Q17*, peptide with sequence Q17RPRPR • Q43, peptide with sequence RPRPRQ43RPRPR • Q38*, peptide with sequence Q38RPRPR • RP-HPLC, reverse phase high pressure liquid chromatography • TFA, trifluoroacetic acid • TIC, Total Ion Current • TG, transglutaminase • tTG, guinea pig liver tissue transglutaminase • Tris, Tris(hydroxymethyl)aminomethane
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Introduction |
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-carboxamide T-group of a peptide-bound glutamine residue and the 
-amino group of a peptide-bound lysine, leading to an isopeptide bond (Folk and Finlayson 1977; Lorand and Conrad 1984). Nine different TG genes have been characterized in mammalian species so far (Grenard et al. 2001); however, the biological significance of some gene products is completely unknown. Although the specialized physiological function of the well-known TGs is to stabilize biological structures via cross-linking of proteins (Aeschlimann and Paulsson 1994; Aeschlimann and Thomazy 2000), recent data indicate that TGs are involved in neurodegenerative disorders such as Qn/CAG repeat disorders, as well as Alzheimer’s and Parkinson’s diseases, and progressive supranuclear palsy (Kim et al. 2002).
Eight hereditary neuropathologies, such as Huntington’s disease (HD), dentatorubral pallidoluysian atrophy, spinobulbar muscular atrophy, and spinocerebellar ataxias type 1, 2, 3, 6, and 7, are caused by the expansion of polyglutamine (Qn) repeats in the encoded proteins (Paulson 1999; Perutz 1999; Zoghbi and Orr 2000). These proteins form insoluble intranuclear and cytoplasmatic aggregates in brains and in animal models of the disease, resulting in neuronal death and progressive neurodegeneration (Davies et al. 1997; DiFiglia et al. 1997; Paulson et al. 1997; Gutekunst et al. 1999; Li et al. 1999). In HD, such aggregates are formed when the expansion of Q residues exceeds a critical number (n > 35–40), leading to a "toxic gain of function" (Perutz 1999). It has been demonstrated that the expanded Qn repeats are potent substrates for tissue TG (tTG) in vitro, resulting in the formation of covalently bonded aggregates with polypeptides that contain lysyl groups (Green 1993; Kahlem et al. 1996; Cooper et al. 1997a) with the rate of cross-linking increasing with the length of the glutamine repeats. It has been suggested that longer Qn stretches not only increase the number of residues potentially available for tTG-mediated cross-linking, but also increase the apparent affinity of glutamines as tTG substrates, thus stimulating tTG activity (Cooper et al. 1999, 2002). Moreover, it was reported that the levels of expression and activity of tTG were increased in the cortex and cerebellum of HD patients (Lesort et al. 1999) simultaneously with significant increased levels of -glutaminyl–lysyl cross-links in nuclear inclusions of HD brain (Karpuj et al. 2002a), as well as in the cerebrospinal fluid of HD patients (Jeitner et al. 2001). Finally, overexpression of the tTG gene in human neuroblastoma cells strongly increases the number and the size of cellular aggregates (de Cristofaro et al. 1999), whereas the inhibition of tTG activity interferes with the accumulation of such cellular inclusions (Karpuj et al. 2002b).
To date, several key cellular proteins interacting with both normal and mutant polyglutamine-stretch-containing disease products have been described (Cha 2000). Such interacting proteins are candidate players in the pathogenesis of Qn disease. In particular, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was shown to tightly bind several proteins involved in Qn-expansion disease: huntingtin, dentatorubral pallidoluysian atrophy protein, ataxin-1, and the androgen receptor in human brain homogenates (Burke et al. 1996; Kosby et al. 1996). In addition, GAPDH was found covalently linked to the Q60 sequence in a Balb-c 3T3 cell line overexpressing human tTG when the cell content was treated with the Q60 polymer (Gentile et al. 1998).
Recently, we have reported the identification of GAPDH reactive lysines in a tTG-catalyzed reaction using Substance P peptide, a known tTG substrate (Esposito et al. 1995), which bears the simplest Qn domain n = 2, (Orrù et al. 2002). K191, K268, and K331 were identified out of the 26 lysines present in GAPDH as the reactive NH2-donor sites, thus indicating a high specificity of the tTG-catalyzed reaction. In the present paper, we describe further studies using synthetic peptides of pathological and nonpathological length (n = 43 and 17, respectively). The aim of the present study is to verify whether tTG-mediated polymerization is a function of the glutamine-repeat length. In addition, we wanted to elucidate whether the different Qn domains form cross-links with the same K residues involved in the presence of Substance P or determine an involvement of different lysines in tTG-catalyzed reactions.
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Two synthetic peptides of pathological and nonpathological length (n=43 and 17, respectively) were designed. The RPRPR sequence was chosen as the flanking region for both peptides in order to increase their solubility (Kahlem et al. 1996). To characterize the two synthetic peptides used in this work, a reverse phase HPLC and a MALDI/MS analysis of the two products were performed separately. The Q17 species was dissolved as reported in a recent study on the solubilization of polyglutamine peptides (Chen and Wetzel 2000; Chen et al. 2001) and analyzed by HPLC. Its chromatographic profile revealed the presence of a very intense peak together with a very minor component. The two peaks were analyzed by MALDI/MS analysis. The mass spectrometric characterization of the major peak showed a unique component at m/z 3522.5, corresponding to the intact Q17 peptide (molecular mass = 3521.8 D). On the other hand, the MALDI/MS characterization of the minor HPLC peak showed a heterogeneous mixture of synthesis fragments with masses <2000 D. Taken together, these results showed that the synthesis of Q17 peptide yielded a >90% pure species.
As previously observed (Altschuler et al. 2000), the Q43 peptide shows a very poor solubility. It was dissolved in a 1:1 HFIP/TFA solution as reported (Chen and Wetzel 2000; Chen et al. 2001). The solubilized peptide was then analyzed by HPLC. The chromatographic profile reveals two main peaks that were characterized by MALDI/MS. Mass data indicated that the first eluting peak corresponded to a broad mixture of synthesis fragments with masses <4000 D, whereas the last eluting HPLC peak contained a polydisperse species having an average molecular mass of 6444 ± 500 D (expected molecular mass of Q43 peptide = 6853.2 D). As might be expected for peptides containing a high number of Q in sequence, the Q43 peptide did not appear to be a pure species, but its Qn sequence ranged mainly from 36 to 43 residues, none of them being predominant.
The Qn peptides were then submitted to an ultraviolet circular dichroism (CD). Q17 was dissolved both in water and in 6 M guanidine. The CD spectrum of Q17 run in water showed that the peptide consists of 25% 
-helix, 32% ß-sheets, and 43% random coil, whereas in the presence of 6 M guanidine, a 100% random-coil conformation was observed (data not shown). A high percentage of random-coil conformation was still monitored in an aqueous solution; however, it should be noted that polyglutamine peptides are able to adopt different conformations when flanked by different sequences (Perutz et al. 1994; Stott et al. 1995; Altschuler et al. 2000; Chen et al. 2001; Masino et al. 2002). CD analysis of the Q43 peptide indicates that it is in a random-coil conformation; however, the polydisperse nature of Q43 makes CD data difficult to analyze. In any case, a detailed structural characterization of Q17 and Q43 using NMR or other techniques was out of the scope of this paper.
Reactivity of Q17 peptide
Q17 was incubated with the amino-donor glycine ethylester (GEE) in the presence of purified tTG. The reaction was monitored in a time-course experiment. The products of the tTG-catalyzed reaction were analyzed by MALDI/MS.
Figure 1
shows MALDI/MS spectra of the aliquots withdrawn at 1, 20, and 120 min. After 1 min, the MALDI spectrum shows the presence of the unmodified peptide together with four other signals corresponding to the Q17 peptide covalently linked to one, two, three, and four molecules of GEE (molecular mass of GEE = 103.6 D). All the assignments are shown in Table 1. After 20 min, the MALDI spectrum shows that up to 12 GEE molecules were bound to Q17 with a distribution centered on the species carrying 7 GEE molecules. After 120 min, up to 16 GEE molecules were found covalently linked to Q17. The reaction profile did not change with prolonged incubation time. It has to be considered that every measured species could consist of a population of isomeric compounds, wherein different Q residues might be involved in the cross-links with the amino-donor substrate. These data hence indicate that all Q17 glutamine residues are sterically accessible and reactive versus a small amino acceptor like GEE.
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Similar experiments were carried out with Q43 peptide to study its reactivity versus GEE or MDC. In both cases, the MALDI spectra showed a very complex mixture in which each component of the polydisperse species had reacted with the amine-donor molecule. Experimental data indicated that GEE had an easy access to the Q43 species, labeling from 30 to 35 Q residues after 120 min of incubation. A lower number of Q residues (20–25) reacted with the sterically hindered amino-donor substrate, thus confirming the trend observed with the Q17 peptide.
Identification of reactive lysine residues of GAPDH in the presence of peptides containing nonpathological Q repeats
GAPDH was incubated with amino-acceptor Q17 peptide in the presence of purified tTG. The mixture formed during the reaction, containing soluble and insoluble products, was hydrolyzed by endoproteinase Asp-N. The proteolytic enzyme produced a suitable digestion of GAPDH, as previously reported (Orrù et al. 2002). The resulting peptide mixture was separated by HPLC coupled on-line with an electrospray mass spectrometer (LCMS).
The Total Ion Current (TIC) profile of the peptide mixture is shown in Figure 2A. Every TIC fraction is related to a mass spectrum. TIC fractions containing the GAPDH peptides modified by Q17 are numbered in Figure 2A. As an example, Figure 2B shows the mass spectrum related to fraction 1. The spectrum shows the presence of a species with mass 5663.4 ± 0.3 D. This signal cannot be assigned to any GAPDH peptide along the protein sequence on the basis of the molecular weight and the specificity of the enzyme. It was interpreted as GAPDH fragments D186–R194 and D323–E332 involved in the formation of a cross-link with a single Q17 molecule via two different Q residues (expected molecular mass = 5664.3 D). Both GAPDH fragments contain a single K residue, K191 and K331, respectively, which were therefore identified unequivocally as tTG amino-donor sites. Table 2 shows the mass signals revealed by LCMS analysis and originated by the formation of cross-links between GAPDH peptides and Q17.
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Fraction 3 contains a species with molecular mass = 5940.5 ± 0.4 D, corresponding to peptide D253–E275 covalently linked to the Q17 peptide; the molecular mass shows the formation of four cross-links involving four different Q residues of the Q17 peptide (expected molecular mass = 5942.6 D). K256, K257, K260, and K268, present in peptide D253–E275, were therefore unequivocally involved in the formation of cross-links.
Fraction 4 contains a species with molecular mass = 10,890.0 ± 0.5 D, which was assigned to GAPDH fragment D241–E275 linked to two Q17 molecules (expected mass = 10,891.1 D). Fragment D241–E275 contains six putative tTG amino-donor sites, K248, K251, K256, K257, K260, and K268, respectively, which could be involved in the formation of cross-links with the two molecules of Q17 peptide.
Finally, fraction 5 contains a species with molecular mass = 7757.0 ± 0.6 D, corresponding to GAPDH fragments D253–C281 and E264–T274 covalently linked with one Q17 peptide (expected molecular mass = 7755.7 D). Peptide D253–C281 contains four putative tTG substrates, K256, K257, K260, and K268; whereas peptide E264–T274 contains a single K residue at position 268, which is therefore involved without ambiguity in the formation of cross-links. This mass signal shows the formation of a dimeric species involving fragments of two different GAPDH molecules covalently linked by the Q17 peptide via two different Q residues.
LCMS analysis was used to unambiguously identify K191, K256, K257, K260, K268, and K331 as tTG substrates and indicated the potential involvement of K248 and K251.
To confirm previous data, the reaction products were also hydrolyzed with trypsin, and the resulting peptide mixture was analyzed by LCMS. In this case, the proteolytic enzyme cleaved both GAPDH and the Q17 peptide. Q17 was hydrolyzed at the level of the first Q in sequence, producing a fragment Q17RPRPR exhibiting a molecular mass of 2859.0 D. The fragment Q17RPRPR was indicated with the abbreviation Q17* in the following text. As a matter of fact, GAPDH fragments linked to Q17* peptide were identified. Modified peptides of GAPDH identified by LCMS analysis are shown in Table 3.
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Fraction 2 contains two species with molecular masses of 5683.4 ± 0.4 D and 5021.2 ± 0.7 D, respectively. The first was assigned to GAPDH fragments L246–K260 and Y252–K260 linked via a cross-link with the Q17* peptide (expected molecular mass = 5681.4 D). Peptide L246–K260 contains four putative tTG substrates, K248, K251, K256, and K257; whereas peptide Y252–K260 contains two putative tTG substrates, K256 and K257. The presence of the cross-links between GAPDH peptides L246–K260 and Y252–K260 shows the formation of a dimeric species involving the fragments of two different GAPDH molecules covalently linked by the Q17* peptide via two different Q residues. The species with molecular mass 5021.2 D contained in fraction 2 was interpreted as GAPDH fragments K105–K114 and K257–K268 linked via a cross-link with the Q17* peptide (expected molecular mass = 5019.4 D). K110, contained in the peptide K105–K114, was unequivocally involved in the formation of the cross-link, whereas K257 and K260, contained in peptide K257–K268, could both act as tTG substrates.
Fraction 3 contains a species with mass = 4107.2 ± 0.4 D, which was assigned to GAPDH fragment K257–K268 covalently linked to Q17* by means of two cross-links involving K257 and K260 (expected mass = 4108.5 D).
Finally, fraction 4 contains a species with molecular mass = 4713.5 ± 0.7 D, which was assigned to GAPDH fragments Y252–K257 and Y252–K260 linked via formation of a cross-link with the Q17* peptide (expected molecular mass = 4715.2 D) involving K256 and K257. The occurrence of this cross-link confirms the presence of a dimeric species involving the fragments of two different GAPDH molecules.
Identification of reactive lysine residues of GAPDH in the presence of peptides containing pathological Q repeats
GAPDH was incubated in the presence of tTG with the Q43 species with the aim of elucidating how the tTG-reactive lysines are affected by a longer Qn stretch. At the end of the reaction, the incubation tube contained a very large amount of insoluble products. The mixture was then submitted to treatment with a 1:1 HFIP/TFA solution as described previously (Chen et al. 2001), in order to improve solubility and to allow the subsequent enzymatic digestion to be more efficient. The treated mixture was submitted to two different extensive hydrolytic steps using endoproteinase Asp-N or trypsin as performed for the sample containing the Q17 peptide, described above. In contrast to what was observed with Substance P (Orrù et al. 2002) and Q17 (see above), the peptide mixtures obtained after the proteolytic digestions were characterized by the occurrence of a conspicuous amount of insoluble pellet. The peptide mixtures were then centrifuged, and the supernatant phases were then analyzed by LCMS. These analyses showed the presence of unmodified GAPDH fragments in the soluble fractions, whereas no signals related to a cross-link formation between the protein and Q43 species were found. The insoluble pellets were again submitted to a further treatment with a 1:1 HFIP/TFA solution in order to improve their solubility. The subsequent LCMS analyses showed the presence of GAPDH peptides modified by Q43. However, they were characterized by a low TIC and very weak mass signals. It was therefore very difficult to obtain reliable data for the identification of GAPDH peptides involved in the formation of cross-links with Q43 peptide. It is also important to note that the Q43 peptide is not a single species but ranges from 36 to 43 residues, as reported before. Only two significant mass signals were observed, shown in Table 4. The peak at molecular mass = 9033.8 ± 0.7 D was assigned to GAPDH fragment D253–T274 (2359.7 D), covalently linked to the species RPRPRQ42RPRPR (6725.1 D). The observed molecular mass showed the occurrence of three cross-links via three different Q residues (expected molecular mass = 9034.8 D). Fragment D253–T274 contains four putative tTG reactive lysine residues, K256, K257, K260, K268. On the basis of the molecular mass, it is possible to state that only three residues out of four are involved in the formation of the cross-links with the polyglutamine peptide. The species at molecular mass = 11,030.0 ± 0.5 D was assigned to GAPDH fragment V160–K212, covalently linked to the species Q38* (expected molecular mass = 11,029.0 D). Q38* resulted from the tryptic hydrolysis of Q38 peptide at the level of the first Q in the sequence producing a fragment Q38RPRPR. Fragment V160–K212 contains two K residues, K183 and K191, one of them being involved in the cross-link. These data show that Q43 reacts with some of the same K residues that are involved in the formation of cross-links with its shorter homologous Q17. However, the extreme insolubility of the samples under investigation only allowed a partial characterization of the cross-links formed in the presence of Q43.
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First, GAPDH was incubated alone under the experimental conditions described above. At the end of the incubation, the solution was centrifuged, and no pellet was observed.
In a second experiment, the Q43 peptide was incubated without tTG and GAPDH under the same conditions. At the end of the incubation, the solution was centrifuged, and the presence of a pellet was observed. The pellet was separated from the supernatant and submitted to treatment with a 1:1 solution of HFIP/TFA. This treatment allowed complete solubilization of the pellet.
Finally, the Q43 peptide was incubated with GAPDH without tTG under the conditions already described. At the end of the incubation, the mixture showed a pellet that was separated from the supernatant. The supernatant was hydrolyzed and characterized by LC/MS analysis as described above. This analysis showed the presence of GAPDH fragments together with signals of Q43 peptide. The pellet was submitted to treatment with a 1:1 solution of HFIP/TFA. This treatment led to a partial solubilization of the pellet, which was still visible in the test tube after a centrifugation step. These data indicate that GAPDH and Q43 form insoluble noncovalent aggregates, which are then cross-linked by the action of tTG (Perutz et al. 1994).
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Recently, K191, K268, and K331 were identified out of the 26 lysines present in GAPDH as the reactive NH2-donor sites in the tTG-catalyzed reaction (Orrù et al. 2002). The present paper reports the characterization of transglutaminase-catalyzed cross-linking between GAPDH and Qn repeats of nonpathological (n = 17) and pathological (n = 43) length. The study confirmed that K191, K268, and K331 are, indeed, reactive NH2-donor sites and led to the identification of K248, K251, K256, K257, and K260 as new electron-donor functionalities in GAPDH. Interestingly, both Qn stretches form cross-links with the same K residues located in the C-terminal region of GAPDH. These results indicate that both Qn stretches interact with the C-terminal domain of GAPDH. The structural characterization reported in this paper showed the occurrence of dimeric species involving different GAPDH molecules. The results indicate that Qn domains can act as a bridge, via different Q residues, between two or more GAPDH molecules, giving rise to diverse multimers.
It is well known that a consensus sequence does not exist around the specific lysine residues acting as amino-donor sites in the tTG-catalyzed reaction; however, it has been suggested that the nature of the amino acid residues directly preceding the lysine may influence its reactivity. Indeed, uncharged, basic, polar, and small aliphatic residues enhance reactivity, which is decreased by residues such as D, G, P, H, and W (Groenen et al. 1994; Grootjans et al. 1995). In this context, the observed reactivity of GAPDH K248, K251, K256, K257, and K260 is in line with this trend. However, Substance P peptide, bearing the simplest Qn domain (n = 2), was not able to recognize the above lysine residues as NH2 donors, despite the fact that they are located in regions with sequences that should have enhanced their reactivity (Orrù et al. 2002). The observation that a higher number of GAPDH K residues are susceptible to cross-linking in the presence of tTG, when using Qn stretches longer than Substance P, indicates that the interaction Qn–GAPDH may cause conformational changes so that GAPDH becomes a better tTG substrate. Because insoluble proteinaceous deposits were formed with Q43 and not with Q17, a preferential interaction of GAPDH and Q43 is indicated. The large formation of aggregates in the presence of pathological-length Qn domains can be explained as a consequence of the peculiar kind of interaction between Qn stretches and proteins. As proposed by Perutz (1999), the expanded Qn repeats may interact with each other through a polar zipper, thus forming a nucleation site for noncovalent interactions in fibrillous aggregates containing Qn stretches and interacting proteins. Such noncovalent aggregates would then become substrates for tTG.
Our findings (Orrù et al. 2002; data presented here) show that all the reactive lysines are present in the C-terminal portion of GAPDH. Therefore, the hypothesis can be put forward that the selective reactivity of the GAPDH lysine residues could be caused by steric hindrance, which is the main factor in the recognition of lysine residues by tTG.
It is important to underline that the catalytic site of GAPDH is localized in the C-terminal domain of the enzyme (Sirover 1999). The fact that all the reactive lysines are located in this domain supports the previous observation that purified GAPDH was inactivated by tTG (Cooper et al. 1997b). This observation indicated that a slow decline in the energy metabolism of neuronal cells may trigger the degenerative process leading to cell death (Burke et al. 1996). Although a decrease in glycolysis seems to be ruled out in HD (Browne et al. 1997), there may be other aspects of GAPDH that are altered in HD brain. In fact, GAPDH has many other functions (apparently as a monomer), and a portion is found in the nucleus, where it appears to have some role in apoptosis (Sirover 1999).
Recently, it has been reported that the inactivation of GAPDH by tTG may contribute other than in HD, also in Alzheimer’s disease (AD; Mazzola and Sirover 2001). In fact, tTG has been implicated not only in the formation of plaques and tangles in AD brain, but also may still play a significant role in AD by participating in apoptosis to cross-link proteins (Citron et al. 2001). Proteins not necessarily belonging to plaques, such as GAPDH and -ketoglutarate dehydrogenase complex, two potential tTG substrates, might contribute to the progression of neurodegenerative disease, being inactivated by tTG in the presence of Q donors (Cooper 1997b). On the basis of the existing overlapping of aggregate composition in different diseases such as HD and AD involving both tTG and GAPDH, it is possible to suggest a key role played by tTG in the specific recognition of GAPDH.
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Materials and methods |
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-cyano--hydroxycinnamic acid, and 1,1,1,3,3,3-hexafluor-2-propanol (HFIP) were purchased from Sigma. Q17 peptide was purchased from Primm, whereas Q43 peptide was purchased from Genemed Biotechnologies, Inc. Endoproteinase Asp-N sequencing grade and trypsin were purchased from Roche Diagnostic GmbH. RP-HPLC columns C18 (25 x 0.21 cm) were purchased from Vydac (The Separation Group). All the other reagents and solvents were of the highest purity available from Carlo Erba. tTG was purified as described previously (Lee et al. 1989).
Chromatographic procedures
Chemically synthetized peptides were purified by performing an RP-HPLC analysis using a C4 Vydac column (25 x 0.46 cm). The eluting system consisted of 0.1% TFA (eluent A) and 0.1% TFA in acetonitrile (eluent B). Both Q17 and Q43 peptides were eluted by means of a linear gradient of eluent B in eluent A, from 5% to 95% in 45 min at a flow rate of 1 mL/min. Elution was monitored at 220 nm.
CD analysis of Q17 and Q43 peptides
Far-UV CD spectra were collected at 25°C on a Jasco J715 spectropolarimeter equipped with a Peltier thermostatic cell holder (Jasco model PTC-348), in a quartz cell of 0.1-cm light path. The temperature was measured directly in the quartz cell. Peptide solutions were filtered just before analysis on a 0.22-µm pore size PVDF membrane (Millipore) and baseline corrected. Spectra were recorded from 250 to 200 nm at 0.2-nm resolution, 16-sec response, at a scan rate of 20 nm/min. All data are the averages of three measurements, and the results are expressed as mean residue ellipticity [
]. Sample concentration was estimated by quantifying the absorbance peak in reverse phase HPLC as already described (Chen et al. 2001)
Reactivity of Q17 peptide
The reactivity of glutamine residues present in the Q17 peptide was evaluated by incubating Q17 peptide with glycine-ethylester (GEE) or with monodansylcadaverine (MDC). Both assays were carried out using 14.1 µg of Q17 and 1.2 µg of GEE (molar ratio Q17/GEE = 1/3) or 4.2 µg of MDC (molar ratio Q17/MDC = 1/3) in the presence of 5 µg of tTG (molar ratio Q17/tTG = 60/1). Assays were carried out in 125 mM Tris-HCl (pH 8.1), containing 10 mM DTT and 2.5 mM CaCl2. Each assay mixture was incubated at 37°C in a total volume of 150 µL. Aliquots were withdrawn at 5, 10, 20, 30, 60, and 120 min. The reactions were stopped by freezing and then lyophilyzing the samples. The mixtures were resuspended in 0.2% TFA and analyzed by MALDI/MS.
Identification of reactive lysine residues of GAPDH in the presence of peptides containing nonpathological and pathological Q repeats
GAPDH was incubated with Q17 and Q43 peptides using 50 µg of the protein and 6.4 µg of Q17 (molar ratio GAPDH/Q17 = 1/10) or 4.4 µg of Q43 (molar ratio GAPDH/Q43 = 1/10) in the presence of 5 µg of tTG (molar ratio GAPDH/tTG = 20/1). The reactions were carried out in 125 mM Tris-HCl (pH 8.1), containing 10 mM DTT and 2.5 mM CaCl2. Each assay mixture was incubated at 37°C for 30 min. The reactions were stopped by freezing and then lyophilyzing the samples.
Enzymatic and nonenzymatic hydrolysis
Samples were resuspended in 50 mM ammonium bicarbonate (pH 8.0). Asp-N hydrolysis was carried out using a 1/100 enzyme-to-substrate ratio (w/w) and 10% acetonitrile. Trypsin hydrolysis was carried out using a 1/50 enzyme-to-substrate ratio (w/w). Reactions were performed at 37°C for 18 h.
Samples deriving from the incubation of GAPDH and Q43 were resuspended in 8 M guanidine and diluited to a 1 M guanidine final concentration with 50 mM ammonium bicarbonate (pH 8.0). The enzymatic hydrolyses were carried out as described above. The resulting peptide mixtures were lyophilyzed and then resuspended with a 1:1 HFIP/TFA solution as previously described (Chen and Wetzel 2000; Chen et al. 2001). The peptide mixtures were then immediately analyzed by LCMS.
Mass spectrometry
MALDI mass spectra were recorded using a Voyager DE mass spectrometer (Applied Biosystem). A mixture of 1 µL of sample solution and 1 µL of -cyano-4-hydroxycinnamic acid (10 mg/mL in acetonitrile/0.2%TFA = 7/3 [v/v]) was applied to the sample plate and allowed to dry. Mass calibration was performed using insulin (average molecular mass = 5734.6 D) and a matrix peak (379.1 D) as internal standard. Raw data were analyzed using computer software provided by the manufacturers and reported as average masses.
Liquid chromatography/mass spectrometry (LCMS) analyses were performed using an LCQ ion trap mass spectrometer (Finnigan Corp.), equipped with an electrospray ion source. The eluting system consisted of 5% formic acid, 0.05% TFA in water (eluent A), and 5% formic acid, 0.05% TFA in acetonitrile (eluent B). The 100-µL aliquots of each peptide mixture were injected onto an RP-HPLC C18 column Vydac (25 x 0.21 cm; The Separation Group) and fractionated by performing a linear gradient of eluent B in eluent A from 5% to 65% B for 60 min, at a flow rate of 200 µL/min. Spectra were acquired from 500 to 2000 D. The resulting mass data were elaborated using the Excalibur software provided by the manufacturer. The mass range was calibrated using an ultramark solution provided by the manufacturer.
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Acknowledgments |
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References |
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