Binding studies on the combining site of a GalNAc alpha 1-->-specific lectin with Thomsen-Friedenreich activity prepared from green marine algae Codium fragile subspecies tomentosoides.
The combining site of a GalNAc alpha 1-->-specific lectin (CFT) with Thomsen-Friedenreich (T, Gal beta 1-->3-GalNAc alpha 1-->Ser/Thr) activity, purified from the subspecies tomentosoides of green marine algae Codium fragile was studied by quantitative precipitin and precipitin-inhibition assays. Of 27 glycoforms tested, Tn (GalNAc alpha 1-->Ser/Thr) glycoprotein from armadillo submandibular glands, and asialo porcine submandibular glycoprotein, which contains T, Tn and GalNAc alpha 1-->3Gal(A) sequences, completely precipitated the lectin added, and less than 1 microgram glycoprotein was required to precipitate 50% 4.7 micrograms lectin nitrogen. However, CFT precipitated negligibly with Pneumococcus type-XIV polysaccharide and asialo human alpha 1-acid glycoprotein, that contain exclusively the human blood-type-II precursor sequence (II, Gal beta 1-->4GlcNAc) at the nonreducing ends. Among the sugar inhibitors tested, the human blood A-active trisaccharide [Ah, GalNAc alpha 1-->3 (LFuc alpha 1-->2)Gal] was the best inhibitor; it was about twice as active as the T disaccharide. Oligosaccharides without GalNAc alpha 1--> as part of their sequences were inactive, indicating that the acetamido group at C2 of galactose is essential for binding and that GalNAc is the main contributor in the T sequence for binding. From the data provided, it is clear that the combining site of CFT requires an alpha-anomer of GalNAc and recognizes Ah, internal GalNAc alpha 1--> of T and Tn determinants of glycans, but not the blood group I/II (Gal beta 1-->3/4GlcNAc) sequences. Consequently, CFT is a useful reagent for detecting GalNAc alpha 1-->-containing glycoconjugates.
Wu
A M
AM
Glyco-Immunochemistry Research Lab., Institute of Molecular and Cellular Biology, Chang-Gung Medical College Tao-yuan, Taiwan.
Song
S C
SC
Hwang
P Y
PY
Wu
J H
JH
Chang
K S
KS
D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D000460:Chlorophyta / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006386:Hemagglutination Tests; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests
D015295:Antigens, Tumor-Associated, Carbohydrate; D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; C016467:Thomsen-Friedenreich antigen; D000116:Acetylgalactosamine