title: Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells.
abstract: Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. 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. The novel 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. 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. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. 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). 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. 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.
authors.affiliation: Graduate Program in Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Promod
authors.initials: P
authors.lastname: Mehndiratta
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Walton
authors.forename: Joan T
authors.initials: JT
authors.lastname: Hare
authors.forename: Silvia
authors.initials: S
authors.lastname: Pulido
authors.forename: Gopalakrishnan
authors.initials: G
authors.lastname: Parthasarathy
authors.forename: Mark R
authors.initials: MR
authors.lastname: Emmett
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Logan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002460:Cell Line; D002645:Chickens / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000235:genetics; D037102:Lectins / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018800:Thy-1 Antigens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019895:Transposases / Q000235:genetics*
chemical_list: D037102:Lectins; D009844:Oligosaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; C473503:Tn5 transposase; D019895:Transposases