PMID: 14711516

 

    Legend: Gene, Sites

Title : Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Abstract :
  1. Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites
  2. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells
  3. The novel isoform of Thy-1 isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues , but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation
  4. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry
  5. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate
  6. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60 , whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5)
  7. The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site
  8. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycoproteins, , glycoproteins, -, -
  • 1. glycosylation, , -, -, sites
  • 3. glycosylation, , -, -, sites
  • 4. N-glycosylation, , Thy-1, -, site
  • 6. Recombinant, , Recombinant Thy-1, 2, -
  • 6. Recombinant, , Recombinant Thy-1, 5, -
  • 6. Recombinant, , Recombinant Thy-1, GlcNAc, -
  • 6. Recombinant, , Recombinant Thy-1, Man, -
  • 6. oligosaccharides, , -, 100, asparagine 23 and 100
  • 6. oligosaccharides, , -, asparagine 23 and 100, asparagine 23 and 100
  • 8. glycoproteins, , -, recombinant glycoproteins, -
  • 8. glycoproteins, , glycoproteins, -, -
Output(Part-Of) (sent_index, protein, site):
  • 4. Thy-1, site
  • 6. Recombinant Thy-1, asparagine 60
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 6. Recombinant Thy-1, 100, asparagine 23 and 100
  • 6. Recombinant Thy-1, asparagine 23 and 100, asparagine 23 and 100

 

 

Protein NCBI ID SENTENCE INDEX
Thy-1 7070 0,2,4,7
isoform of Thy-1 378897 3
Lec1 23266 0,2,6
Recombinant Thy-1 7070 6
form of Thy-1 7070 2