β-Aspartyl Peptides in Enzymatic Hydrolysates of Protein<sup>*</sup>

Haley, Edward E.; Corcoran, Betty J.; Dorer, Frederic E.; Buchanan, Donald L.

doi:10.1021/bi00874a024

Cited by 63 publications

(22 citation statements)

References 23 publications

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“…In a previous study, Haley et al (40) were not able to show an increase in the number of ␤-isomerized Asp-Gly sequences from Achilles tendon per molecule of type I collagen with increasing age. In the same study, Haley et al (40) estimated the content of ␤-isomerized Asp-Gly sites to be between 0.8 and 1.9 per collagen molecule.…”

Section: ␤-Isomerization Of Type I Collagenmentioning

confidence: 78%

Characterization of Urinary Degradation Products Derived from Type I Collagen

Fledelius¹,

Johnsen

Cloos³

et al. 1997

Journal of Biological Chemistry

183

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The heterogeneity of urinary degradation products of C-terminal telopeptides derived from the ␣1 chain of human type I collagen was investigated and characterized. The urinary fragments characterized in this study consisted of two cross-linked (X) amino acid sequences derived from the C-terminal telopeptide (␣1) of type I collagen. Fragments containing the sequence EXAH-DGGR, with a DG site being either nonisomerized (AspGly) or ␤-isomerized (␤Asp-Gly), were identified. Pyridinoline was detected among the pyridinium crosslinks, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that of galactosyl pyridinoline. A nonfluorescent cross-link was also found. The concentration of fragments derived from the C-terminal telopeptide region of type I collagen containing the sequence Asp-Gly (␣CTX) and/or ␤Asp-Gly (␤CTX) was measured by enzyme-linked immunosorbent assays in urine and in collagenase digests of trabecular and cortical bone of young and old origin. It was shown that the urinary ratio between such fragments, ␣CTX/␤CTX, was higher in children compared with adults and that the ratio decreased with increasing age of bone. The results indicated that the C-terminal telopeptide fragments derived from type I collagen excreted into urine originated mainly from bone. In conclusion, it is demonstrated for the first time that the C-terminal telopeptide ␣1 chain of type I collagen contains an Asp-Gly site prone to undergo ␤-isomerization and that the degree of ␤-isomerization of this linkage apparently increases with increasing age of bone. These findings indicate that the ratio ␣CTX/␤CTX might be clinically important in diagnosing metabolic bone diseases.The rate of bone turnover has for years been estimated by measurement of different markers reflecting bone formation and bone resorption in urine and blood (1). The traditional bone-resorption markers are urinary calcium and hydroxyproline. Hydroxyproline, however, is not specific for bone (2), and its urinary excretion is influenced by diets rich in hydroxyproline (3), renal clearance, and metabolization (4, 5).The pyridinium cross-links pyridinoline (Pyr) 1 and deoxypyridinoline (Dpyr) are trifunctional cross-links that together with other di-, tri-, and tetrafunctional cross-links stabilize the collagen structure within the extracellular matrix (6). Pyr is found mainly in bone and cartilage (7,8) but also in collagen from most other connective tissues, whereas Dpyr is present mainly in bone and dentin (7). Although the pyridinium cross-links are not entirely bone-specific, they appear to be superior markers of bone resorption compared with hydroxyproline (1). The pyridinium cross-links occur at two intermolecular sites, one linking two C-terminal telopeptides to residue 87 of the triple helix ␣1 or ␣2 chain and the other linking two N-terminal telopeptides to residue 930 of the triple helix ␣1 chain (9). The C-terminal telopeptide ␣1 chain of type I collagen consists of a 26-amino acid sequen...

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Section: ␤-Isomerization Of Type I Collagenmentioning

confidence: 78%

Characterization of Urinary Degradation Products Derived from Type I Collagen

Fledelius¹,

Johnsen

Cloos³

et al. 1997

Journal of Biological Chemistry

183

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“…Isoaspartyl linkages themselves are generally not cleaved by mammalian proteases (33,34). We have thus investigated the possibility that proteolysis of proteins containing damaged aspartyl residues in PCMT1-deficient mice may be reflected in the increased urinary output of peptides containing L-isoaspartyl residues.…”

Section: Discussionmentioning

confidence: 99%

“…Few peptidases cleave isoaspartyl bonds, but proteolysis of the surrounding residues creates isoaspartyl-containing dipeptides and tripeptides that can be excreted in the urine (33)(34)(35)(36). Thus, if mice lacking endogenous PCMT1 do not have another repair pathway, they might excrete the damaged residues that are normally repaired in the cells of wild-type mice.…”

Section: Accumulation Of Damaged Aspartyl Residues In Brainmentioning

confidence: 99%

Limited Accumulation of Damaged Proteins inl-Isoaspartyl (d-Aspartyl)O-Methyltransferase-deficient Mice

Lowenson¹,

Kim

Young

et al. 2001

Journal of Biological Chemistry

137

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L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1)can initiate the conversion of damaged aspartyl and asparaginyl residues to normal L-aspartyl residues. Mice lacking this enzyme (Pcmt1؊/؊ mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1؊/؊ mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1؊/؊ mice lived, on average, 5-fold longer than nontransgenic Pcmt1؊/؊ mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concentration of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1؊/؊ mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1؊/؊ mice contained increased amounts of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.The spontaneous chemical modification of proteins by reaction with oxygen, water, sugars, and other abundant metabolites is unavoidable. The accumulation of such nonenzymatically altered proteins is associated with normal aging as well as atherosclerosis, Alzheimer's disease, and diabetes (1-3). Organisms have several strategies for dealing with damaged proteins, including intracellular proteolysis mediated by proteasome and lysosome action (4 -6). Some types of covalent damage, however, are simple enough to recognize and repair directly (7). Enzymes such as prolyl cis-trans isomerase (8), methionine sulfoxide reductase (9), and disulfide isomerase (8) can restore activity to proteins that have been chemically altered.We are interested in a common type of spontaneous protein damage in which L-aspartyl and L-asparaginyl residues undergo an intramolecular reaction that converts them to L-succinimidyl residues (10, 11). Nonenzymatic hydrolysis of the succinimide ring readily occurs at either carbonyl to generate both normal aspartyl residues and isoaspartyl residues, in which the peptide backbone proceeds through the ␤-carbonyl rather than the ␣-carbonyl moiety (12). The succinimide also racemizes more rapidly than do the open chain forms, and hydrolysis of the D-succinimide produces D-aspartyl and D-isoaspartyl residues (12). Local protein structure causes some L-aspartyl and L-asparaginyl residues to be especially prone to succinimide formation, and the presence of damaged aspartyl residues at these sites can significantly alter the structure, function...

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“…The first method is based on the interruption of Edman sequencing of a polypeptide at the isomerized derivative, due to the failure of phenylthiohydantoin formation in the acid-cleavage step of the Edman degradation reaction (e.g., see Takio et al, 1986). The second method is based on the insusceptibility of the fi-linkage aspartyl to proteolytic digestion, with consequent formation and analysis of small isomerized peptides that can be compared to standard peptides with known fl-isomerized linkages (e.g., see Haley et al, 1966;and Aswad et al, 1988). The third method involves the ability of protein D-aspartyl/L-isoaspartyl carboxylmethyltransferase to specifically methylate fl-isomerized e-aspartic acid residues in a radiometric methylation assay (Ota and Clarke, 1990;Johnson and Aswald, 1990).…”

Section: Discussionmentioning

confidence: 99%

Determination of Β-isomerized aspartic acid as the corresponding alcohol

Carter

McFadden

1994

J Protein Chem

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The age-related formation of succinimides in proteins, through spontaneous deamidation of asparagine, and through cyclization of aspartic acid, is thought to be followed by the hydrolysis of the succinimide ring, yielding a mixture of "normal" aspartic acid sites and beta-isomerized aspartic acid sites (isoaspartic acid). The chemical reduction of an isoaspartyl site to the corresponding amino acid alcohol, isohomoserine, has now been investigated as a general approach to measuring the accumulation of isomerized residues in aging proteins. The methods employed were based on conditions previously found to be successful in reducing protein aspartic acid to homoserine. Borane was employed as the reducing agent, and was found to produce the expected amino acid alcohols in reactions with model peptides. In addition, amino acid analysis revealed a complex pattern of unknown products of these reduction reactions, some of which were also evident when a much stronger reducing agent, lithium aluminum hydride, was used. The correlation of some of these side-products with the isomerization of the peptide suggests, unexpectedly, that the reactivity of reducing agents toward aspartyl residues and perhaps other sites in the peptide may be influenced by steric factors related to aspartyl isomerization. The borane reduction method was also applied to proteins. No detectable isohomoserine was formed either in ovalbumin, a model aged protein, or in human lens proteins of advanced age, with conditions that fully reduced normal aspartyl residues to homoserine. These tests thus indicate that the percentage of aspartic acid in the isomerized form in these proteins is below the limit of detectability (below approximately 5%). These results complement previous experimental results that have indicated a low bulk isoaspartyl content in most natural proteins.

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β-Aspartyl Peptides in Enzymatic Hydrolysates of Protein^*

Cited by 63 publications

References 23 publications

Characterization of Urinary Degradation Products Derived from Type I Collagen

Characterization of Urinary Degradation Products Derived from Type I Collagen

Limited Accumulation of Damaged Proteins inl-Isoaspartyl (d-Aspartyl)O-Methyltransferase-deficient Mice

Determination of Β-isomerized aspartic acid as the corresponding alcohol

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β-Aspartyl Peptides in Enzymatic Hydrolysates of Protein*

Cited by 63 publications

References 23 publications

Characterization of Urinary Degradation Products Derived from Type I Collagen

Characterization of Urinary Degradation Products Derived from Type I Collagen

Limited Accumulation of Damaged Proteins inl-Isoaspartyl (d-Aspartyl)O-Methyltransferase-deficient Mice

Determination of Β-isomerized aspartic acid as the corresponding alcohol

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β-Aspartyl Peptides in Enzymatic Hydrolysates of Protein^*