Analysis of human ocular mucus: effects of neuraminidase and chitinase enzymes.
OBJECTIVE
Our goal was to establish the characteristic migration pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of high molecular weight mucins from human ocular mucus and the effects of treatment with exo- and endoglycosidases.


METHODS
Chromatography by gel filtration with Sepharose CL-4B was performed on samples collected from normal subjects. Human ocular mucins from the high molecular weight fraction were digested with exoglycosidases (neuraminidase, N-acetyl-beta-D-glucosaminidase, beta-D-glucosidase) and endoglycosidases (chitinase, lysozyme); and the resulting products were analyzed by electrophoresis. Carbohydrate identification was performed using lectin probes.


RESULTS
The migration of the ocular mucins on SDS-PAGE stopped after treatment with neuraminidase, which removes the terminal negatively charged sialic acid residues from mucin. Chitinase (beta(1-4)N-acetylglucosaminidase) treatment increased the electrophoretic migration of mucins. Staining with wheat germ agglutinin and Maackia amurensis agglutinin lectins showed that these mucins contain beta(1-4)NAcGlc and SAa(2-3)Gal linkages.


CONCLUSIONS
These studies demonstrate that the mobility of human ocular mucins on SDS-PAGE is determined by their intrinsic total negative charge and is not dependent on SDS treatment. It is interesting to note that human ocular mucus contains chitinous material resistant to lacrimal lysozyme, which is accessible to chitinase, an enzyme now found to degrade human ocular mucins. These chitinous linkages could be in part responsible for the mucus resistance.
Argüeso
P
P
Instituto de Oftalmobiología Aplicada, and Department of Biochemistry and Molecular Biology, Sciences School, University of Vallodolid, Spain.
Herreras
J M
JM
Calonge
M
M
Citores
L
L
Pastor
J C
JC
Girbés
T
T
D000293:Adolescent; D000328:Adult; D002688:Chitinases / Q000494:pharmacology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007765:Lacrimal Apparatus / Q000737:chemistry*; D037102:Lectins; D008297:Male; D008875:Middle Aged; D009077:Mucins / Q000032:analysis*; D009093:Mucus / Q000737:chemistry* / Q000187:drug effects; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009439:Neuraminidase / Q000494:pharmacology*; D011134:Polysaccharides / Q000032:analysis; D012685:Sepharose / Q000031:analogs & derivatives
D037102:Lectins; D009077:Mucins; D011134:Polysaccharides; C035771:Sepharose CL 4B; D012685:Sepharose; D002688:Chitinases; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid