Higher glycation of beta L- and beta S-crystallins in the anterior lens cortex and maximum glycation of gamma-crystallins in the bovine lens nucleus, demonstrated by frozen sectioning, isoelectric focusing and lectin staining.
The aim of the current study was to demonstrate glycation of beta L-, beta S- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into alpha-crystallins of high molecular weight (HM), alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. In the lectin staining experiments, only HM, beta L-, beta S- and gamma-crystallins were positive, whereas the alpha L- and beta H-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta S- and beta L-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total gamma-crystallins 2.44, beta S-crystallins 0.77 and beta L-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of gamma-crystallins was 3 times higher than that of beta S-crystallins and 9 times higher than that of beta L-crystallins.
Bours
J
J
Institute of Experimental Ophthalmology, University of Bonn, Germany.
Ahrend
M H
MH
Utikal
K J
KJ
D000818:Animals; D002417:Cattle; D003208:Concanavalin A; D003459:Crystallins / Q000378:metabolism*; D003720:Densitometry; D005629:Frozen Sections; D006031:Glycosylation; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007904:Lens Cortex, Crystalline / Q000378:metabolism*; D007907:Lens Nucleus, Crystalline / Q000378:metabolism*; D012394:Rosaniline Dyes; D013194:Staining and Labeling
D003459:Crystallins; D007202:Indicators and Reagents; D012394:Rosaniline Dyes; D003208:Concanavalin A; C048139:Coomassie blue