Identification of glucose-derived cross-linking sites in ribonuclease A.
The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Delta m/ z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins.
Dai
Zhenyu
Z
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Wang
Benlian
B
Sun
Gang
G
Fan
Xingjun
X
Anderson
Vernon E
VE
Monnier
Vincent M
VM
D000595:Amino Acid Sequence; D005947:Glucose / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D012259:Ribonuclease, Pancreatic; D005947:Glucose