PMID: PMC7108604-1-3

 

    Legend: Gene, Sites

Title : Liberation and fractionation of glycans

Abstract :

1. Western blot analysis, using a VE-cadherin monoclonal antibody, of endothelial cells treated with PNGase F and endo-β-N-acetylglucosaminidase H (endo H) revealed the presence of both endo H-sensitive and endo H-resistant N-glycans
2. Treatment with PNGase F resulted in a shift of the VE-cadherin band from about 135 kDa to about 90 kDa (arrow) in agreement with the expected molecular mass calculated from its amino acid sequence (Figure 2, lane 3)
3. The second band (dotted line) is assumed to represent a degradation product
4. Incubation with endo H also led to a small but significant shift in the electrophoretic mobility of this glycoprotein (Figure 2, lane 5)
5. Therefore, isolated glycopeptides were first treated with endo H. Oligosaccharides released were separated from residual glycopeptides by reversephase (RP-) HPLC
6. Endo H-resistant glycopeptides were incubated with PNGase F
7. The resulting reaction mixture was again subjected to RP-HPLC to isolate free glycans
8. Approximately 16% and 73% of total radioactivity were released by the two enzymes
9. Residual 11% of radioactivity, still eluting in the peptide fraction, could be shown to comprise exclusively N-acetylglucosamine (GlcNAc) and no N-acetylgalactosamine (GalNAc) and are, therefore, assumed to result from the innermost GlcNAc residue (s) remaining bound to the peptide(s) after endo H cleavage (Kobata, 1979)
10. After reduction, oligosaccharide alditols were fractionated by anion-exchange HPLC
11. The results revealed that the majority of human VE-cadherin glycans carry negative charges
12. Complex type species, released by PNGase F , comprised neutral glycans (F0, 7% of radioactivity) in addition to species with one (F1, 49%), two (F2, 38%), or three (F3, 5%) negatively charged residues (Figure 3A)
13. Oligosaccharides released by endo H, representing oligomannosidic or hybrid-type glycans (Kobata, 1979), similarly carried predominantly (75%) one negative charge (data not shown)
14. Treatment with sialidase from V.cholerae or mild acid hydrolysis converted all charged species into neutral compounds demonstrating that the negative charge was exclusively conferred by sialic acid
15. In the case of PNGase F-sensitive oligosaccharide alditols, charged glycans were separated by preparative anion-exchange HPLC and individually digested with α2,3-specific sialidase from Newcastle disease virus
16. Monosialylated species could not be degraded and, thus, solely contained α2,6-linked sialic acid , whereas di- and trisialylated glycans both contained one (or two) α2,6-linked sialic acid residue (s) in addition to α2,3-bound sialic acid (Figure 3B–D)
17. For further characterization, all glycans were completely desialylated

Output (sent_index, trigger, protein, sugar, site):

• 12. one, , -, -, residues
• 4. glycoprotein, , glycoprotein, -, -
• 5. glycopeptides, , -, -, glycopeptides
• 6. glycopeptides, , -, -, glycopeptides

Output(Part-Of) (sent_index, protein, site):

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX