Identification of lectin binding proteins in human tears.
The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.
Kuizenga
A
A
Biochemical Laboratory, Netherlands Ophthalmic Research Institute, Amsterdam.
van Haeringen
N J
NJ
Kijlstra
A
A
D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007071:Immunoglobulin A, Secretory / Q000032:analysis; D007781:Lactoferrin / Q000032:analysis; D037102:Lectins / Q000378:metabolism*; D008970:Molecular Weight; D016622:Silver Staining; D013666:Tears / Q000737:chemistry*
D005136:Eye Proteins; D006023:Glycoproteins; D007071:Immunoglobulin A, Secretory; D037102:Lectins; C042434:tear proteins; D007781:Lactoferrin