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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. These results indicate that the lectin-like domain of rhLT is exposed on its desialylation.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Fukushima K,Watanabe H,Takeo K,Nomura M,Asahi T,Yamashita K

doi

10.1006/abbi.1993.1332

subject

Has Abstract

pub_date

1993-07-01 00:00:00

pages

144-53

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(83)71332-9

journal_volume

304

pub_type

杂志文章

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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.