Presence of D-aspartate and D-glutamate in tumor proteins.

Over 50 years ago Kögl and Erxleben reported that tumor proteins contain appreciable amounts of D-amino acids. This postulate remained highly controversial for several years, during which time several researchers either supported or refuted the hypothesis. We have analyzed several sets of tumors and normal control tissues for the presence of D-aspartate (D-Asp) and D-glutamate (D-Glu). Most tumors contain less D-Asp than the control tissues, whereas nearly half of the tumors contain 1.6 to 5.4 times more D-Glu than the controls. The tumors averaged 0.72% D-Asp and 0.61% D-Glu compared to 0.94% D-Asp and 0.35% D-Glu in the control tissues. However, within the limits of experimental error, there is no significant difference in the level of these D-amino acids between the tumors and normal tissues.

Altered aspartate in Alzheimer neurofibrillary tangles.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo post-translational modification by racemization to D-aspartate, or by isomerization to the L-isoaspartyl form in which the peptide chain links through the beta carboxyl group of the residue. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a significantly greater number of these modified aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Racemized D-aspartate in Alzheimer neurofibrillary tangles.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo posttranslational modification by racemization to D-aspartate. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a greater number of these racemized D-aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Free D-aspartate and D-alanine in normal and Alzheimer brain.

In this report we present evidence for the presence of free D-aspartic acid (D-Asp) and D-alanine (D-Ala) in the white and gray matter of normal human brains and brains of individuals with Alzheimer's disease. D-Asp occurs at about the same concentration in the gray matter of both normal (18.6 nmol/g) and Alzheimer (14.8 nmol/g) brains, whereas in white matter its concentration is more than two times higher in normal than Alzheimer brains (22.4 and 10.5 nmol/g, respectively). D-Ala occurs in white matter at approximately the same concentration in both normal and Alzheimer brains (12.3 and 13.8 nmol/g, respectively), whereas in Alzheimer gray matter the D-Ala concentration is more than twice that found in normal gray matter (20.8 and 9.5 nmol/g, respectively). However, when the results are expressed as a percentage of D-amino acid/D+L, only small differences occur in all tissues examined.

T lymphocytes can recognize determinants unique to neuropeptides of guinea pig myelin basic protein containing a single D-isomer amino acid substitution.

The present studies were undertaken to examine how the substitution of racemized forms of selected amino acids in synthetic peptides of guinea pig myelin basic protein (GPMBP) would alter the host's immunological ability to recognize such molecules. Using peptides from the 69-84 sequence of GPMBP containing a D-serine at position 70 or 75 (69-84[D-ser70 or D-ser75]) or D-aspartate at position 82 (69-84[D-asp82]), the findings demonstrated that the position of the diastereomer substitution on these neuropeptides was critical with respect to the ability of the immune system to recognize the molecule. Thus substitution of D-asp at position 82 or D-ser at position 75 abrogated the ability of these peptides to induce experimental autoimmune encephalitis and proliferation of host T cells. In contrast, a peptide containing a D-ser70 residue was capable of inducing clinical disease in rats, as well as stimulating T lymphocytes from 69-84-(D-ser70)-injected animals. Moreover, although this D-peptide was shown to share at least some determinant(s) with the 69-84 peptide, the use of 69-84(D-ser70)-stimulated cell lines demonstrated that some epitope(s) unique to this molecule could stimulate CD4+ syngeneic T cells.

D-aspartate in human brain.

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.

D-aspartic acid in purified myelin and myelin basic protein.

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.

Analysis of problems encountered in the determination of amino acid enantiomeric ratios by gas chromatography.

A previously described procedure for determining the enantiomeric ratios of amino acids has produced inconsistent results when determining relatively low (less than or equal to 0.110) D/L ratios. The method involves synthesis of diastereomeric N-trifluoroacetyl-L-prolyl-D/L-amino acid ester dipeptides which are resolved by gas chromatography (GC). We have found that triethylamine, which is added to maintain a basic pH during the coupling reaction, racemizes the chiral reagent N-trifluoroacetyl-L-prolyl chloride (TPC). Coupling of partially racemized TPC to D/L-amino acid esters results in the formation of four dipeptides (two pairs of enantiomers) instead of the expected two diastereomeric dipeptides. The enantiomeric dipeptides coelute on an achiral GC column, resulting in erroneous D/L ratios. More accurate D/L ratios are obtained by preparing the volatile N-trifluoroacetyl-D/L-amino acid isopropyl ester derivative which can be separated into its enantiomers on a chiral GC column such as the Chirasil-Val III (registered trademark of Applied Science Laboratories).

Accumulation of D-aspartic acid with age in the human brain.

An age-related accumulation of D-aspartic acid was detected in the white matter of ten normal brains from individuals aged 30 to 80 years. Gray matter showed no systematic increase in D-aspartic acid. The rate constant for D-aspartate formation in the brain is equal to the predicted value calculated for 37 degrees C. Accumulation of the uncommon D-aspartate isomer in myelinated white matter implies that there is little or no turnover of this tissue, and this may have a bearing on dysfunction of the aging brain or on other diseases of myelin.