The objective of the work described here was to examine and compare both i) protein carbonylation in general and ii) carbonylation at specific amino acid residues in a protein as a function of increasing OS using diabetic and lean animals.
The blood proteome was chosen as a sample source because of the ease with which it could be obtained and still provide oxidized proteins originating from multiple organs. At the general level involving the assay of all carbonylated isoforms of proteins together by Protocol A, the data shows clearly that there is no relationship between protein abundance in plasma and protein carbonylation. Although oxidized serum albumin was found, this highly abundant protein was not a major contributor to the carbonylated plasma proteome. Abundant proteins in plasma are either not as easily oxidized or carbonylation occurs at local sites and oxidized proteins are exported into plasma. The later seems the most likely based on the fact that so many proteins in plasma are of tissue origin. This makes localized OS a major source of carbonylated proteins in plasma. Compartmentalization seems to be important in protein carbonylation. It also explains why the level of some oxidized proteins in plasma is elevated more than others. They experience differing levels of localized OS in the compartments where they reside.
Actually the importance of the location of proteins at the time of oxidation and the source of oxidants has been reported in the case of intestinal fatty acid binding protein (I-FABP). This protein plays an important role in the transport of fatty acids to mitochondria and their subsequent β-oxidation.40
The close proximity of I-FABP to mitochondria and fatty acids provides a potential explanation for how malondialdehyde and methylglyoxal adducts are formed in diabetic rats. A previous study has shown that carbonylation of A-FABP (adipocyte FABP) with 4-HNE is elevated in vivo
in adipose tissue of insulin resistant mice.41
Hemopexin, clusterin and Apo AII were observed to have experienced the greatest increases in carbonylation among plasma proteins. Presumably this is due to elevated ROS levels having oxidized a greater proportion of these proteins in diabetic rats versus lean controls but increased expression might have been a factor as well. Hemopexin42
expression has been found to increase in the plasma of type II diabetes. Another study showed that increased expression of Apo AII was a factor in insulin resistance.44
A 1.5 fold increase in oxidized protein isoforms has been shown to be strongly correlated with kidney and coronary heart diseases. Whether oxidized proteins cause or are the result of these phenomena remains to be determined. A study quantifying both changes in protein expression and carbonylation is needed to clarify the mechanism of how the elevation of these proteins occurred in diabetics.
With specific proteins data from these studies shows that i) that oxidation is specific and reproducible, and ii) that the extent of oxidation at any one site as a function of increasing OS is quantitatively independent of that at other sites. Some sites are dramatically more labile than others as seen in the cases of hemoglobin, murinoglobulin and fibrinogen. There is also no relationship between the mole fraction of carbonylation across all sites in a protein and changes at individual sites. It’s currently impossible to detect all the carbonylation sites of a protein therefore; the net change of all oxidized isoforms of a protein can’t be correlated to the oxidation levels at different sites.
One of the most distinguishing features of protein carbonylation was that oxidation patterns were protein specific in terms of i) the dominant oxidation mechanism, ii) the amino acids being modified, and iii) the oxidation mechanisms involved.
With hemoglobin for example, OS induced post-translational modifications by the ALE mechanism were elevated 20 fold in diabetic subjects relative to lean controls at residue K69 (by HNE addition) and 2 fold at residue K12 (with malondialdehyde addition). Deoxyglucosone modification at reside K49 was the same in lean and diabetic subjects. It is surprising that in a diabetic subject with elevated glucose levels and accelerated ROS production that carbonylation by glucose induced AGE mechanisms was the same as in lean controls. The dramatic increase in carbonylation from lipid peroxidation in contrast is expected based on the elevation of OS in diabetics. The presence of iron in hemoglobin could also increases the potential for oxidation, as would be the case with other metal containing proteins such as serotransferrin, ceruplasmin, hemopexin, and fibrinogen.45
Changes in AGE based carbonylation were equally dramatic with murinoglobulin 1 homolog. Although there was no significant change in total carbonylation at all sites, carbonylation by AGE mechanisms at residues K347 and K352 decreased 100 fold in diabetic subjects while ALE based malondialdehyde adduct formation and direct carbonylation at residues 682 and 731 respectively were unchanged. Again, a decline in carbonylation by the AGE mechanism when ROS and glycation are increasing with diabetic progression is not expected in a diabetic. Why ALE based carbonylation at residues K69 and K12 in hemoglobin was substantially elevated in diabetics while there was a significant reduction in AGE base adduction formation at residue K347 and K352 in murinoglobin 1 is a mystery.
Enhanced degradation of oxidized proteins could explain the selective decrease of some oxidized proteins. Previous reports indicated that there is an increase in proteolysis of some oxidized and glycated proteins in the plasma of diabetic patients, particularly in the case of methyl glyoxal adducts.49 Moreover, the activity of oxidized protein hydrolase (OPH) in the serum of diabetic rats increased nearly in parallel to the increase of blood glucose levels.50 In addition, oxidized proteins are ubiquitinated and form aggregates in the cytoplasm of pancreatic β cells that degrade through autophagy.51 Diabetes induced OS in the retinal endothelial cells and up-regulated the ubiquitin proteasomal system in yet another study.52
Fibrinogen alpha polypeptide isoform 1 showed still another pattern, there was no significant change in total carbonylation at all sites while arginine oxidation in diabetic subjects increased 2 fold at residue 419 and 6 fold at residue 770, respectively.
Protein conformation along with changes in tertiary structure seems to be another factor in protein oxidation. A recent study showed that glyceraldehyde-3-phosphate dehydrogenase changes its conformation in vivo
according to the degree of OS.46
With this protein, oxidatively induced changes in conformation increase the probability of oxidation at other sites.