Chemical modification of arginine residues of lung galaptin and fibronectin. Effects on fibroblast binding.
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ABSTRACT:
SUBMITTER: Powell JT
PROVIDER: S-EPMC1152970 | biostudies-other | 1985 Dec
REPOSITORIES: biostudies-other
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Project description:Modification of arginine residues using dicarbonyl compounds is a common method to identify functional or reactive arginine residues in proteins. Arginine undergoes several kinds of posttranslational modifications in these functional residues. Identifying these reactive residues confidently in a protein or large-scale samples is a very challenging task. Several dicarbonyl compounds have been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione. However, tracking these reactive arginine residues in a protein or large-scale protein samples using a chemical labeling approach is very challenging. Thus, the enrichment of modified peptides will provide reduced sample complexity and confident mass-spectrometric data analysis. To pinpoint arginine-labeled peptide efficiently, we developed a novel arginine-selective enrichment reagent. For the first time, we conjugated an azide tag in a widely used dicarbonyl compound cyclohexanedione. This provided us the ability to enrich modified peptides using a bio-orthogonal click chemistry and the biotin-avidin affinity chromatography. We evaluated the reagent in several standard peptides and proteins. Three standard peptides, bradykinin, substance P, and neurotensin, were labeled with this cyclohexanedione-azide reagent. Click labeling of modified peptides was tested by spiking the peptides in a myoglobin protein digest. A protein, RNase A, was also labeled with the reagent, and after click chemistry and biotin-avidin affinity chromatography, we identified two selective arginine residues. We believe this strategy will be an efficient way for identifying functional and reactive arginine residues in a protein or protein mixtures.
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Project description:Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-?3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to ?(V)?(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.
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