Terminal marking of triosephosphate isomerase: consequences of deamidation.

Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

Terminal marking of avian triosephosphate isomerases by deamidation and oxidation.

Triosephosphate isomerase (TPI) provides an excellent model for terminal marking and protein degradation. Mammalian TPI is terminally modified by deamidation at Asn71-Gly, resulting in unfolding, dissociation, and proteolysis. In contrast, chicken TPI, which contains a lysine at position 71, is terminally modified by the oxidation of Cys126. Thus, both deamidation and oxidation initiate degradation of TPI from different species. To explore the terminal marking in other avians, we have purified the turkey TPI to homogeneity and determined its characteristics. Although the molecular properties of the turkey and chicken TPI were very similar, their tolerances to temperature, oxidants, and alkaline pH were very different. For example, chicken TPI was inactivated 80% in either 10 mM oxidized glutathione or H2O2, whereas 120 mM GSSG had no effect on turkey TPI, and > 120 mM H2O2 was needed for 80% inactivation. Under alkaline conditions that cause rapid deamidation of the mammalian TPI, neither avian enzyme deamidated. Chicken TPI, however, aggregated. Aggregation was reversed by 2-mercaptoethanol. Under prolonged exposure to milder conditions the turkey enzyme was completely inactivated and deamidated at Asn15-Gly. Thus, there are marked differences in the susceptibility of these two avian enzymes to oxidation and deamidation, and their terminal marking mechanisms appear to be different.

Deamidation of triosephosphate isomerase in reverse micelles: effects of water on catalysis and molecular wear and tear.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

The hinged lid of yeast triose-phosphate isomerase. Determination of the energy barrier between the two conformations.

Covalent modification of Glu165 in the catalytic center of triose-phosphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting 31P NMR resonances at 6.9 and 5.7 ppm appear to reflect conformations in which the hinged lid (residues 167-176) is in the open and closed positions. The conformation represented by the 5.7-ppm resonance is more stable, and unfolding and refolding in guanidine converts all of the molecules to the 5.7-ppm conformation. The complete conformational transition from 6.9 to 5.7 ppm also takes place as a function of time and temperature. Under these conditions the native enzyme retains more than 80% of the catalytic activity, indicating that this conversion is not due to thermal denaturation of the enzyme. Circular dichroic and fluorescence spectroscopy indicate that the 3-chloroacetol phosphate-modified enzyme does not undergo major structural changes. From the temperature dependence of this transition, an energy barrier of 144 kJ/mol (34.4 kcal/mol) was calculated for this conversion.

Limited proteolysis of triose-phosphate isomerase and characterization of the catalytically active peptide complex.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.

Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.

Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

Cellular models and tissue equivalent systems for evaluating the structures and significance of age-modified proteins.

The accumulation of modified proteins in aging is well documented in many aging models. For example, the deamidated isoforms of triosephosphate isomerase accumulate in: (a) old erythrocytes, (b) fibroblasts from old donors, (c) fibroblasts aged in vitro, (d) premature-aging syndromes and (e) old cells in the eye lens. However, a fundamental remaining question is: 'Do such modified proteins interfere with cellular function?' It has been difficult to assess this question at the molecular level using whole-organism models and equally frustrating to evaluate the physiological significance of such changes using classical cellular models. Tissue equivalent systems (TES) provide an opportunity for examining the molecular basis and physiological consequences of modified proteins during aging. TES are composed of differentiating and proliferating heterogeneous cell types with symbiotic cell-cell and cell-matrix interactions. They closely resemble, both morphologically and functionally, the tissues from which they were derived. Aging studies utilizing TES can provide information on modifications of protein structures, isozyme patterns, enzymes of the cellular environmental protection system and metabolic parameters which may regulate protein synthesis and degradation.

Isoforms of chicken triosephosphate isomerase are due to specific oxidation of cysteine126.

The electrophoretic isoforms of mammalian triosephosphate isomerase (TPI; EC 5.3.1.1) are due to deamidation at two Asn-Gly sites (Asn15 and Asn71). Deamidation of these two asparagines in the subunit-subunit interface of the isologous dimer appears to destabilize the dimer and initiate degradation of the protein. Chicken TPI contains a lysine substitution for Asn71, thus precluding this deamidation site. Nevertheless, the chicken enzyme exhibits three electrophoretic isoforms. This multiplicity is not the result of deamidation of the remaining Asn15 site, but due to a specific site which is highly susceptible to oxidation. The three isoforms of chicken TPI can be reduced to a single form in the presence of high concentrations of reducing agents (e.g., greater than 15 mM dithiothreitol or greater than 50 mM 2-mercaptoethanol) and are also generated when oxidizing agents, such as oxidized glutathione, are present. The oxidized isoforms exhibit lowered catalytic activity and are more susceptible to denaturation and proteolytic degradation than the native enzyme. Structural analysis of the isoforms by chemical cleavage at the cysteine peptide bonds with 2-nitro-5-thiocyanobenzoic acid and subsequently at the methionines with CNBr followed by peptide sequencing reveals that Cys126 is the site of the modification. Since the oxidized isoforms of chicken TPI accumulate in vivo during aging analogous to the deamidated isoforms from mammals, it appears that TPI is the first example of a protein which has evolved two specific types of weak links which may initiate turnover of the protein.

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits.

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.

The accumulation of oxidized isoforms of chicken triosephosphate isomerase during aging and development.

Triosephosphate isomerase (TPI) from mammals undergoes two specific deamidations (Asn-15 and Asn-71) which destabilize the isologous dimer and lead to the degradation of the protein. In aging cells and tissues, the deamidated isoforms accumulate apparently due to age-related changes in protein turnover. Chicken TPI lacks one of these sites (i.e., Asn 71----Lys), but also exhibits unstable isoforms. These isoforms are the result of the specific oxidations which occur both in vitro and in vivo. Electrophoretic analyses of various tissues from chicken show that the most oxidized isoform, which is present in adult tissues, is only present in small quantities in tissues of the newborn chick. Moreover, embryonic tissues contain almost exclusively the fully reduced form of TPI. Thus, it appears that oxidation rather than deamidation constitutes the first step in the degradation of avian TPI. TPI may be the first example of a protein which has evolved two different types of modifications (deamidation and oxidation) which trigger its degradation. The accumulation of both deamidated and oxidized isoforms in different species may provide clues to the underlying basis for the accumulation of modified proteins in aging.

Rapid isolation of triosephosphate isomerase utilizing high-performance liquid chromatography.

A new method for the isolation of homogeneous triosephosphate isomerase (TPI, EC 5.3.1.1) has been developed. The method utilizes high-performance liquid chromatography on DEAE 5PW and Hydrophase-polyethyleneimine columns, which results in the rapid isolation and essentially quantitative recovery of the enzyme. The procedure is superior to previous methods with respect to specificity, recovery, and time. In addition, this rapid process minimizes the potential for postsynthetic modifications of the protein. Milligram quantities of TPI can be isolated from 100 g of tissue.

Effects of aging and xerosis on the amino acid composition of human skin.

Amino acid compositions of skin samples from young and old subjects and from age-matched donors with dry skin syndrome (xerosis) were examined. The amino acid contents of the free amino acid (FAA) fraction, soluble hydrolysate (SH) fraction, and whole cell hydrolysate (WCH) were determined. The greatest differences were observed between the FAA compositions of the young and old normal subjects. Xerosis did not appear to affect the amino acid compositions of samples from young subjects as much as old subjects. Overall, the effect of aging on the amino acid contents was more pronounced than the effect of xerosis. The amino acid composition of the FAA showed a high degree of similarity to filaggrin, whereas the WCH showed a similarity to keratin.

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes.

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens.

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).

In vitro deamidation of human triosephosphate isomerase.

The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo.