Title :
N-linked sugar chain structure of recombinant human
lymphotoxin produced by CHO cells: the functional role of
carbohydrate as to its lectin-like character and clearance velocity
Abstract :
- Recombinant human lymphotoxin ( rhLT ) produced by CHO cells transfected with human LT genomic DNA was purified to homogeneity, but approximately 5% of the molecules were devoid of the last two amino terminal residues
- A peptide N-glycosylated at Asn62 (Tr-45) and one partially O-glycosylated at Thr7 (Tr-14) on cleavage with trypsin were separated by reverse phase HPLC
- The N-linked sugar chains of Tr-45 were released quantitatively as oligosaccharides on hydrazinolysis (100 degrees C, 8 h), followed by N-acetylation
- After being reduced with either NaB3H4 or NaB2H4, their structures were determined by a combination of serial lectin affinity chromatography, exoglycosidase digestion, and methylation analysis: 82.7% of the sugar chains occur as biantennary complex-type sugar chains , the remainder being C-2 and C-2 ,4/ C-2 ,6 branched triantennary, and C-2 ,4 and C-2,6 branched tetraantennary complex-type sugar chains with a fucosylated mannose core.
- Their sialic acid residues occur only as the Neu5Ac alpha 2-->3Gal group.
- The clearance velocity from the bloodstream dramatically increased with desialylation , and rhLT tends to have accumulated in the kidney, indicating that there may exist other mechanisms for clearance from the circulation besides the galactose-binding protein in hepatocytes and the filtration system of the kidney
- Desialylated rhLT showed a lectin-like binding character to uromodulin similar to that of tumor necrosis factor , although intact rhLT did not
- The interaction between desialylated rhLT and uromodulin was inhibited by N,N'-diacetylchitobiose and [Man alpha 1-->6(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc-->Asn. Asn
- These results indicate that the lectin-like domain of rhLT is exposed on its desialylation