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Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase

¹ Dipartimento di Chimica, Università di Salerno, Salerno, Italy
² Centro di Studio di Biocristallografia, CNR, Naples, Italy
³ Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Naples, Italy

Reprint requests to: Prof. Carla Esposito, Dipartimento di Chimica, Università di Salerno, Via S. Allende, 84081 Baronissi, Salerno, Italy; e-mail: esposito{at}mbox.chem.unisa.it; fax:39-089-965296.

(RECEIVED May 9, 2001; FINAL REVISION September 17, 2001; ACCEPTED October 10, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.17102.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross-links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine-containing peptide was used as probe for labeling the amino-donor sites. SDS gel electrophoresis of a time-course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino-donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino-donor residues modified by tissue transglutaminase.

Keywords: GAPDH; lysine residues; mass spectrometry; tissue transglutaminase

Abbreviations: Asp-N, endoproteinase Asp-N • DNS, dansyl group, ESI-MS, electrospray mass spectrometry • HD, Huntington disease • LC/MS, liquid chromatography/mass spectrometry • MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry • Qn, polyglutamine repeat containing n Q residues • RP-HPLC, reverse phase high pressure liquid chromatography • SubP, substance P • TG, transglutaminase • TIC, total ion current • tTG, guinea pig liver tissue transglutaminase

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Transglutaminases (TGs, EC 2.3.2.13) are calcium-dependent enzymes that catalyze an acyl transfer reaction between the -carboxamide 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). This reaction results in cross-linked protein polymers that are resistant to chemical and enzymatic attacks. Proteolytic enzymes able to hydrolyze these cross-links have not been identified in vertebrates (Melino and Piacentini 1998). At present, distinct widely expressed TG molecular forms have been described, including plasma factor XIIIa, keratinocyte TG (type I), tissue TG (type II, tTG), epidermal TG (type III), prostate TG (type IV; Aeschlimann and Paulsson 1994) and TG X (Kim et al. 1999). TG enzymes are involved in many fundamental biological processes including the fibrin clotting cascade (Folk and Finlayson 1977), seminal vesicle coagulation (Esposito et al. 1996), cornification of the epidermis, hair, and nails (Steinert et al. 1999), extracellular matrix and bone formation (Aeschlimann and Thomazy 2000), apoptosis (Melino and Piacentini 1998). Although the physiological function of these enzymes is to stabilize biological structures, there is increasing evidence that tTGs may play a role in the pathology of neurodegenerative diseases associated with (CAG)_n expansion in the genome and to corresponding polyglutamine (Q_n) repeats in the encoded protein that characterizes the disorder (Paulson 1999; Perutz 1999). There is a lot of evidence indicating that a common feature of the progressive neurodegenerative disorders is the accumulation of Q_n-containing protein aggregates described in the nucleus and in the cytoplasm of affected neurons of patients, in mice models of CAG disease, and in cell-based systems (Davies et al. 1997; DiFiglia et al. 1997; Paulson et al. 1997; Gutekunst et al. 1999; Li et al. 1999). Several clinical and experimental findings suggest that aggregates are formed when the expansion of Q residues exceeds a critical number (n > 35–40), leading to a toxic gain of function that causes cell-specific neurodegeneration (Perutz 1999). The expanded Q_n repeats may interact with each other through a polar zipper, thus contributing to aggregate formation (Perutz et al. 1994). On the other hand, it has been shown that proteins with Q_n repeats are substrates of tTG, resulting in the formation of covalently bonded aggregates with polypeptides that contain lysyl residues (Green 1993; Kahlem et al. 1996; Cooper et al. 1997a,1999). These findings are supported by the observation that overexpression of the TG gene in human neuroblastoma cells greatly increase the number and size of cellular aggregates, whereas the inhibition of TG activity interferes with the accumulation of such cellular inclusions (de Cristofaro et al. 1999).

Kahlem and coworkers showed that the brain possesses K donor–containing proteins able to form covalent bridges with Q_n domains (Kahlem et al. 1996). Subsequently, the glycolytic enzyme GAPDH was shown to tightly bind several proteins involved in Q_n expansion disease: huntingtin, atrophin-1, ataxin-1, and the androgen receptor (Burke et al. 1996; Kosby et al. 1996) in human brain homogenates. 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). These findings suggested that GAPDH possesses at least one reactive K residue per monomer forming bridges between two or more Q_n stretches (Cooper et al. 1999). To date, no structural data are available to solve the issue of how many and which lysine residues in GAPDH are substrates for TG enzyme.

The work described in the present article shows that rabbit muscle GAPDH is a lysyl donor substrate of the guinea pig liver tTG. GAPDH was incubated in the presence of tTG and a Q-donor peptide, Substance P (SubP), which previously was shown to be a substrate of tTG (Esposito et al. 1995). The products of the reaction of GAPDH and SubP in the presence of tTG were characterized by SDS-PAGE and Western blot analyses. The reactive amino-donor sites were identified by mass spectrometry. This article reports that only three lysines, of the 26 lysine residues present in GAPDH, are involved in the formation of cross-links with SubP. These results suggest a high specificity of the tTG-catalyzed reaction.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Glyceraldehyde 3-phosphate dehydrogenase is a substrate of tissue transglutaminase
GAPDH was incubated with dansylated amino-acceptor Substance P (RPKPQQFFGLM) (DNS-SubP) in the presence of purified tTG. The reaction was conducted for different time lengths after a time-course experiment (1, 4, and 18 h). The formation of insoluble products, clearly visible in the assay tubes, forced us to set up a reaction mixture for each incubation period, within a single experiment. The products of the tTG-catalyzed reaction were analyzed by 12% SDS-PAGE, fluorescence, and Western blot analyses.

The Coomassie-stained SDS-PAGE pattern shown in Figure 1A revealed the presence of the GAPDH monomer migrating at 36 kD in the reaction mixtures after 1, 4, and 18 h of incubation (Fig. 1A, lanes 4, 5, and 6, respectively). At the same time, very high molecular weight species appeared from the first stages of reaction. Such polymers hardly penetrated the gel mesh for short reaction times (Fig. 1A, lane 4) whereas for longer periods of incubation they could not leave loading gel wells because of their very large size (Fig. 1A, lanes 5,6). The formation of polymers was inhibited by the addition of a calcium chelator (data not shown). Polymers were also present when SubP was omitted from the reaction (Fig. 1A, lane 3). These results confirm that GAPDH contains reactive glutamines (Ikura et al. 1998) and lysines (Cooper et al. 1997a; Gentile et al. 1998) because intermolecular cross-links are formed in the presence of tTG.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Substrate capacity of GAPDH in tTG-mediated cross-links. SDS-PAGE gel Coomassie blue stained (A), fluorescence (B) and corresponding Western Blot analysis performed using anti-tTG antibodies (C) of the in vitro incubation of rabbit muscle GAPDH (50 µg) with dansylated SubP (18 µg) in the presence of purified tTG (5 µg) at 37°C for 1, 4, and 18 h (lanes 4, 5, and 6, respectively). GAPDH and purified tTG were used as controls (lanes 1 and 2, respectively). As additional control, GAPDH was incubated at 37°C for 18 h in the presence of tTG (lane 3). Protein marker levels are indicated.

Western blot analysis performed using anti tTG antibodies (Fig. 1C) showed an intense band at a high molecular weight. This finding reveals that tTG takes part in the formation of high molecular weight heteropolymers together with DNS-SubP and GAPDH.

Identification of reactive lysine residues by mass spectrometric analysis
The SDS-PAGE bands at 36 kD (Fig. 1A, lanes 4–6) were excised and separately submitted to an in-gel hydrolysis to identify the K residues involved in the covalent cross-links with DNS-SubP. An involvement of GAPDH Q residues in the formation of cross-links was not expected because the K residue in SubP was reported previously not to be a tTG substrate (Porta et al. 1988).

The proteolytic step was conducted on each band, using endoproteinase Asp-N sequencing grade, and the resulting peptide mixtures were analyzed by MALDI-MS. The proteolytic enzyme was chosen because of its ability to hydrolyze GAPDH and to produce a suitable peptide mixture, leaving the DNS-SubP intact. The reasoning behind this choice was to facilitate the assignment of peptides linked to intact DNS-SubP. As a control, the amino acid sequence of GAPDH was characterized using the same strategy as described in Materials and Methods.

MALDI-MS spectra of the peptide mixtures, produced by the hydrolysis of the incubation product at 1, 4, and 18 h, showed signals that could be assigned to GAPDH fragments on the basis of their molecular weights and the specificity of the proteolytic enzyme. Only a few mass signals could not be related to peptides along the GAPDH sequence. However, they were interpreted as GAPDH fragments covalently linked to DNS-SubP via an isopeptide bond. Figure 2 shows a partial MALDI-MS spectrum of the peptide mixture obtained from the hydrolysis of the assay after 1 h. Signals at m/z 2105.1, 2489.8, 2604.8, 3397.6, and 3821.2 were assigned to peptides along the GAPDH sequence on the basis of their molecular weights. An additional signal at m/z 3878.5 was detected and assigned to peptide A312-K331 covalently linked to DNS-SubP (expected m/z 3878.2). The sequence of the identified GAPDH fragment meant K331 could be identified unequivocally as a tTG amino-donor site. Table 1 shows the mass signals due to the formation of cross-links between GAPDH peptides and DNS-SubP at different incubation times. The signals at m/z 3879.0 and 3879.9 were also observed at 4 and 18 h, thus confirming the occurrence of a cross-link involving K331 as an amino donor. An additional signal at m/z 2579.8 was detected in the 4-h mixture and assigned to peptide D186-R194 linked to DNS-SubP (expected m/z 2579.7). Peptide D186-R194 contains a single K residue at position 191, which therefore is involved in the cross-link formation. The presence of this signal only after 4 h seems to suggest that K191 is less reactive than K331. The absence of the signal 2579.7 after a longer incubation time (18 h) can be caused by low recovery of the peptides extracted from the gel. The characterization of reactive lysines in solution (see below) showed, in fact, the presence of this signal also at 18 h.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Partial MALDI-MS spectrum of the peptide mixture derived by Asp-N hydrolysis of the 1-h incubation mixture. The assignment of peaks to the corresponding peptides is shown.

Finally, it is important to consider that the peptide mixtures contained both modified and unmodified forms of the GAPDH fragments, indicating that the selective enzymatic modification of lysine residues is not complete, that is, some GAPDH molecules contain unmodified K residues whereas others contain the same residues modified by tTG catalyzed reaction.

To confirm the previous data, the 4-h mixture was separated by HPLC using a C18 reverse phase column and analyzed on-line by electrospray mass spectrometry (ESI-MS). The total ion current (TIC) profile of the peptide mixture is reported in Figure 3A. Every TIC fraction is correlated to a mass spectrum; the analysis of each mass spectrum provided unambiguous confirmation that K191, K268, and K331 are tTG substrates as shown by previous experiments. The TIC fractions containing GAPDH peptides modified by SubP are numbered in Figure 3A. Figure 3B shows the mass spectrum correlated to fraction 1 as an example. The mass spectrum shows the presence of three different peptides, one of them being the fragment E264-E275 covalently linked to SubP molecule.

View larger version (22K): [in this window] [in a new window]   Fig. 3. (A) LC/ESI-MS analysis of the peptide mixture obtained from endoproteinase Asp-N hydrolysis of the 4-h incubation mixture. Fractions containing modified GAPDH peptides are numbered. Peptides present in the numbered fractions are shown. Peptides containing cross-links with SubP are shown in boldface. (B) Transformed ES mass spectrum of fraction 1. Assignments of the mass signals to the corresponding peptides are shown.

Localization of lysine residues in the three-dimensional structure of GAPDH
Over the years, several crystallographic structures of GAPDH from different sources have been reported. All these models share a high structural similarity. In fact, GAPDH is a tetrameric enzyme the subunits of which have a cofactor binding site and a catalytic domain. To identify the position of the lysine residues in the three-dimensional structure of rabbit GAPDH, we used the structure of human skeletal muscle GAPDH (Mercer et al. 1976) as a model, because the sequences of rabbit and human enzymes are very similar (90% of the residues are identical). Molecular graphics analysis shows that, with the sole exception of K306 located at the intersubunit interface, all the lysines are solvent exposed (Fig. 4). This observation is in agreement with the results obtained by Lambert and Perham (1977) who showed that only one amino group per subunit of rabbit GAPDH was completely unavailable for the reaction with methyl acetamidate.

View larger version (61K): [in this window] [in a new window]   Fig. 4. GAPDH tetramer drawn using the coordinates of the Protein Data Bank entry code 3GDP (Mercer et al. 1976). A ribbon representation has been used to draw one subunit. The balls represent the location of lysine residues in this subunit. The number of the reactive lysine residues (in black) refers to the human sequence. The number of reactive lysine residues along the rabbit sequence is given in parenthesis. The trace of the other subunits is also shown. The figure was drawn using MOLSCRIPT (Kraulis 1991).

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
The accumulation of glutamine-repeat containing protein aggregates within the cytosol and nuclei of affected neurons appears to be a crucial step in the pathogenesis of progressive neurodegenerative disorders, such as Huntington disease (HD;Davies et al. 1997; DiFiglia et al. 1997; Paulson et al. 1997; Gutekunst et al. 1999; Li et al. 1999). Nuclei from dying neurons in the basal ganglia and the brain cortex of transgenic animals with the mutated huntingtin gene have been shown to contain large, insoluble high molecular protein aggregates (Davies et al. 1997). It has been reported that polyglutamine-expanded proteins are able to associate both to themselves and to specific proteins. For example, huntingtin associates with polymerized microtubules in vitro (Tukamoto et al. 1997), and ataxin-1 binds to leucine-rich acidic nuclear protein (Matilla et al. 1997). It has been hypothesized that a polar zipper model serves as a nucleation site for noncovalent interactions in fibrillous aggregate formation of huntingtin and other proteins (Perutz et al. 1994; Liu et al. 2001). The same author, who initially put forward the hypothesis that huntingtin aggregation could take place through the polar zipper action (Perutz et al. 1994), more recently (Perutz 1999) suggested that aggregate formations also could occur through cross-links catalyzed by tTG or by a combination of both mechanisms. In fact, it was shown that polyglutamine domains are good substrates of the enzyme tTG in vitro, and that larger pathological-length Q_n domains are the most active substrates of the enzyme (Cooper et al. 1997a).

At present, there is little information concerning the amino-donor partner of polyglutamine containing proteins to form covalently bonded aggregates (Kahlem et al. 1996; Cooper et al. 2000). It has been shown that the glycolytic enzyme GAPDH binds tightly to several disease proteins such as huntingtin, atrophin-1, ataxin-1, and the androgen receptor (Burke et al. 1996; Kosby et al. 1996). The finding that four different proteins, implicated in different neurodegenerative diseases, interact with GAPDH suggests that common biochemical mechanisms involving GAPDH may lead to different pathogenesis.

On the basis this, we have undertaken a study aimed at structurally characterizing the products of the tTG catalyzed reaction involving GAPDH. We showed that GAPDH is a lysyl donor substrate of tTG in vitro. Surprisingly, of 26 lysines present in the GAPDH sequence (Lambert and Perham 1977), only K191, K268, and K331 are sites of tTG-dependent cross-link formation. In principle, the observed specificity could be for several possible reasons: (1) the sequence around the NH₂ donor, (2) the solvent accessibility of lysine side chains, and (3) steric hindrance between tTG and GAPDH, which may prevent the tTG recognition of specific lysine residues.

It is well known that it has not been possible to derive a consensus sequence around the specific lysine residues from the few substrate proteins in which the amino-donor sites have been identified (Pucci et al. 1988; Porta et al. 1991; Mariniello et al. 1993; Grootjans et al. 1995). Nevertheless, it has been shown 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 (Grootjans et al. 1995). In this context, the observed reactivity of GAPDH K331, which is preceded by a serine residue, is in line with this trend (Groenen et al. 1994; Grootjans et al. 1995). The reactivity of K331 also is enhanced by its location in the C-terminal end of the protein. These results support the general pattern seen so far for other substrate proteins that essentially head and tail domain sequences participate in cross-link reactions (Steinert et al. 1999).

The reactivity of K268, located in the L-K-G sequence, is not surprising because it has been shown that a leucine residue directly adjacent to the lysine substrate has a positive effect on its reactivity (Groenen et al. 1994). The reactivity of GAPDH K191, which is preceded by a glycine, is certainly surprising, as it has been shown that this residue has an adverse effect on the tTG reaction (Grootjans et al. 1995). To the best of our knowledge, this is the first observed case in which a K residue, preceded by a glycine residue, turns out to be a tTG substrate. However, note that other GAPDH lysine residues are not amino donors, despite the fact that they are located in regions with sequences that should have enhanced their reactivity. Altogether, these observations show that previous data on the effect of the local structure on lysine reactivity can only partially explain the specificity of the cross-linked site of GAPDH.

The analysis of the three-dimensional model of GAPDH suggests that 25 of 26 lysines are solvent exposed, and therefore they can be potential amino donors. In fact, it has been reported that these lysines are reactive toward chemical reagents (Lambert and Perham 1977). In this scenario, the selectivity of the GAPDH lysine residues in the reaction catalyzed by tTG could be mostly caused by steric hindrance, which rules the recognition of lysine residues by tTG.

The catalytic site of GAPDH is localized in the C-terminal region (Sirover 1999). Our findings that all the reactive lysines are present in the C-terminal portion of the molecule may explain the previous observation that purified GAPDH was inactivated by tTG in the presence of glutathione S-transferase constructs containing a Q_n domain of pathological length (Cooper et al. 1997b). This observation suggested that loss of GAPDH activity might contribute to the decrease of energy metabolism in HD brain (Burke et al. 1996). However, GAPDH is expressed in enormous amounts relative to huntingtin, and it is improbable that binding of huntingtin to GAPDH could make much difference to the level of the glycolytic enzyme. Conversely, GAPDH, which was studied for more than 3 decades for its pivotal role in glycolysis, has multifunctional activities and diverse subcellular localizations. These include roles for GAPDH in membrane transport and fusion, microtubule assembly, DNA replication, and DNA repair (Sirover 1999). In addition, GAPDH may play a role in apoptosis, which specifically involves its translocation to the nucleus (Saunder et al. 1997; Sawa et al. 1997). Sequence analysis suggests the possibility that amino acids within the catalytic domain, containing the tTG reactive lysine residues, may be involved in the regulation of the subcellular localization of GAPDH in mammalian cells (Yoneda 1997). Therefore, it is conceivable that TG-catalyzed post-translational modification of GAPDH may influence its nuclear translocation as well as any of its other several functions.

In conclusion, we set up an easy and reliable strategy for the identification of tTG-reactive lysine residues in GAPDH. Although we used SubP that has no involvement in HD pathology, the strategy can be effectively used to study the aggregation processes with extended polyQ domains containing normal and pathological Q repeats.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Materials
Rabbit muscle GAPDH, guinea pig liver tissue transglutaminase (tTG), Substance P (SubP), NAD⁺, dithiothreitol (DTT), -cyano-4-hydroxycinnamic acid, and SDS-PAGE protein standards were purchased from Sigma. Iodoacetamide (IANH₂) was obtained from Fluka and endoproteinase Asp-N sequencing grade from Roche Diagnostics GmbH. RP-HPLC columns C18 (25 x 0.46 cm) and (25 x 0.21 cm) were purchased from Vydac (The Separations Group). All reagents for electrophoresis application were purchased from Bio-Rad. Pipette Zip-Tip C₁₈ for sample preparation were obtained from Millipore. 1-Dimethylamino naftalen solphonil chloride (dansylchloride, DNS-Cl) and all other reagents and solvents were of the highest purity available from Carlo Erba.

tTG was purified as previously described (Lee et al. 1989).

Structural characterization of GAPDH
An aliquot of GAPDH corresponding to 35 µg (1 nmole) was denatured in 6 M Guanidine-HCl, 0.25 M Tris-HCl, 1.25 mM EDTA at pH 8.5 and reduced with DTT using a reagent molar excess of 10 : 1 versus cysteine moles. The reaction was performed for 2 h at 37°C. The reduced and denatured GAPDH then was alkylated by incubation with iodoacetamide. The alkylating agent was added to the protein solution using a -SH/IANH₂ = 1 : 10 molar ratio. The reaction was conducted at room temperature for 1 h and then halted by RP-HPLC desalting. Chromatographic analysis was performed using a Vydac C₄ column (25 x 0.46 cm) and an elution system consisting of 0.1% TFA (eluent A) and 0.07% TFA in 95% acetonitrile (eluent B). The modified protein was eluted by means of a linear gradient of eluent B from 10% to 80% over 40 min, at a flow rate of 1 mL/min. Elution was monitored at 220 nm, and the collected fractions were analyzed by ESI-MS. The fraction containing the alkylated GAPDH was lyophilized and then dissolved in 50 mM ammonium bicarbonate at pH 8.1. The protein then was submitted to enzymatic hydrolysis using endoproteinase Asp-N. The reaction was performed for 18 h at 37°C and stopped by adding 5 µL of 5% TFA. The peptide mixture was analyzed by MALDI-MS, and mass signals were assigned to GAPDH fragments on the basis of their molecular weights and the specificity of the enzyme.

The structural analysis meant that the whole sequence of GAPDH from rabbit muscle could be verified. Note that it was possible to identify the amino acid at position 123 as alanine, reported as an X in the SWISS-PROT sequence (accession number P46406). This experimental evidence is supported by the finding of Ala 123 in the GAPDH sequence from several other sources such as pig muscle (Harris and Perham 1968), lobster muscle (Davidson et al. 1967), ox liver (Heinz and Kulbe 1970), and Bacillus stearothermophilus (Walker et al. 1980).

Synthesis of DNS-SubP
Substance P (100 µg) was dissolved in 100 µL of 0.2 M NaHCO₃ at pH 8.8 and incubated with 200 µL of a 5-mg/mL DNS-Cl acetone solution. The reaction was performed in the dark at 37°C. After 3 h, when the starting yellow mixture turned colorless, the reaction was stopped and the DNS-SubP was purified by RP-HPLC analysis using a C₁₈ column (25 x 0.46 cm). The DNS-SubP was eluted using the same eluents described above, by means of a linear gradient of solvent B from 5% to 95% over 60 min, at a flow rate of 1 mL/min. Elution was monitored at 220 nm, and individual collected fractions were identified by ESI-MS.

tTG-catalyzed modification of lysine residues
Modification of K residues in GAPDH was performed by incubating 50 µg of the protein with 18 µg of SubP (molar ratio GAPDH/SubP = 1 : 10) in the presence of 5 µg of tTG (molar ratio GAPDH/tTG = 20 /: 1) in 125 mM Tris-HCl at pH 8.1, 10 mM DTT, 2.5 mM CaCl₂ (final volume 32 µL). The experiments performed in the presence of NAD⁺ were set up by adding a GAPDH/NAD⁺ = 1 : 1 molar ratio. Each assay mixture was incubated for 5 min, 30 min, 1 h, 4 h, or 18 h at 37°C. Eighteen-hour incubations were provided with an additional 5-µg aliquot of tTG after 4 h. The reactions were stopped by lowering the pH (5 µL of TFA 5%) followed by lyophilization. Similar experiments were performed using DNS-SubP and then characterized by means of electrophoresis analyses (SDS-PAGE, fluorescence assay and Western blot analysis).

SDS-PAGE
The lyophilized samples were resuspended in 15 µL of sample buffer and analyzed by SDS-PAGE. SDS gel electrophoresis was performed on 12% linear slab gels as described by Laemmli (Laemmli 1970). Protein bands were stained by means of a Coomassie Blue Brilliant treatment. Molecular masses of protein bands were estimated by comparing their electrophoretical mobility to that of the molecular weight markers.

Fluorescence assay
The assay was performed on the samples analyzed by SDS-PAGE. Samples were incubated in a solution containing 50 mM Tris-HCl at pH 7.1, 9 M urea, 2% SDS, 40 mM DTT for 1 h before loading the mixtures on the stacking gel wells. After the electrophoresis run, the gel was rinsed in the dark in a 10% acetic acid, 25% 2-propanol solution for 15 min. Afterward, a picture of the gel was taken in the dark using a transilluminator (Tulp 20 M; Genenco).

Western blot
Protein bands were electroblotted onto a nitrocellulose strip. Binding of primary antibody, directed against tTG, was revealed using horseradish peroxidase-conjugate goat anti-rabbit IgG and 4-chloro-1-naphtol/hydroxyperoxide as chromogenic agents.

Enzymatic hydrolysis
Lyophilized samples were resuspended in 50 mM ammonium bicarbonate at pH 8.0. Enzymatic digestions with endoproteinase Asp-N were performed using a 1 : 100 enzyme-to-substrate ratio (w/w). Acetonitrile (10%) was added to activate the enzyme. Reactions were conducted for 18 h at 37°C and stopped by adding 5% TFA.

In-gel digestions of proteins were performed on the Coomassie blue–stained protein-containing bands excised from the gel. Gel pieces were washed with acetonitrile and then with 0.1 M ammonium bicarbonate. Protein samples were reduced by incubation in 10 mM DTT for 45 min at 56°C and alkylated with 55 mM iodoacetamide in 0.1 M ammonium bicarbonate for 30 min at room temperature, in the dark, as previously described (Esposito et al. 2001). The gel particles were then washed with ammonium bicarbonate and acetonitrile. Enzymatic digestions were conducted with endoproteinase Asp-N sequencing grade 15 ng/µL in 50 mM ammonium bicarbonate at pH 8.5 at 4°C for 4 h. The enzyme/buffer solution then was removed and a new aliquot of the buffer solution was added for 18 h at 37°C. A minimum reaction volume sufficient for complete rehydration of the gel was used. Peptides then were extracted washing the gel particles with 20 mM ammonium bicarbonate and 0.1% TFA in 50% acetonitrile at room temperature and then lyophilized.

Each peptide mixture was analyzed by MALDI-MS. In some cases, the samples were previously desalted using C₁₈-Zip-Tip from Millipore.

Mass spectrometry
MALDI mass spectra were recorded using a PerSeptive Biosystem Voyager DE Instrument. 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 Daltons) and a matrix peak (379.1 Daltons) as internal standards. Raw data were analyzed using computer software provided by the manufactures and reported as average masses.

ESI-MS analyses were conducted using a Bio-Q triple quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ion source. Samples were injected into the ion source (kept at 80°C) at a flow rate of 5µL/min using a syringe pump Harvard Apparatus mod. 11. Data were acquired and elaborated using the Masslynx program, purchased from Micromass. Mass calibration was performed by means of multiply charged ions from a separate injection of horse heart myoglobin (Sigma; average molecular mass: 16951.5 Daltons); all masses are reported as average values.

LC/MS analysis was performed using an API100 mass spectrometer (PerSeptive Biosystem) equipped with an electrospray ion source. Thirty microliters peptide mixture was injected onto a RP-HPLC C₁₈ column (25 x 0.21 cm) and fractionated by performing a linear gradient from 5% to 65%B in 60 min, at a flow rate of 200 µL/min. The eluate then was split 1/5 before entering into the ion source. Spectra were acquired from 450 to 2000 Daltons. The resulting mass data were elaborated using the BioMultiview software provided by the manufacturer. The mass range was calibrated using horse heart myoglobin.

	Acknowledgments

 
Dedicated to the memory of our friend and colleague Guido Sodano, Professor of Organic Chemistry. This work was supported by CNR grant CNRG0072CF, Progetto Giovani-Agenzia 2000.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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