Novel fluorescent glycan microarray strategy reveals ligands for galectins.
Galectin-1 (Gal-1) and galectin-3 (Gal-3) are widely expressed galectins with immunoregulatory functions in animals. To explore their glycan specificity, we developed microarrays of naturally occurring glycans using a bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB), directly conjugated through its arylamine group by reductive amination to free glycans to form glycan-AEABs (GAEABs). Glycans from natural sources were used to prepare over 200 GAEABs, which were purified by multidimensional high-pressure liquid chromatography and covalently immobilized onto N-hydroxysuccinimide-activated glass slides via their free alkylamine. Fluorescence-based screening demonstrated that Gal-1 recognizes a wide variety of complex N-glycans, whereas Gal-3 primarily recognizes poly-N-acetyllactosamine-containing glycans independent of N-glycan presentation. GAEABs provide a general solution to glycan microarray preparation from natural sources for defining the specificity of glycan-binding proteins.
Song
Xuezheng
X
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Xia
Baoyun
B
Stowell
Sean R
SR
Lasanajak
Yi
Y
Smith
David F
DF
Cummings
Richard D
RD
D000818:Animals; D001549:Benzamides / Q000138:chemical synthesis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D037483:Galectin 1 / Q000378:metabolism; D037502:Galectin 3 / Q000378:metabolism; D037161:Galectins / Q000378:metabolism*; D008024:Ligands; D046228:Microarray Analysis; D011134:Polysaccharides / Q000138:chemical synthesis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C539333:2-amino-N-(2-aminoethyl)benzamide; D001549:Benzamides; D005456:Fluorescent Dyes; D037483:Galectin 1; D037502:Galectin 3; D037161:Galectins; D008024:Ligands; D011134:Polysaccharides