Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone α-subunit

Abstract

Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common alpha- and a specific beta-subunit. A recombinant baculovirus containing a cDNA encoding the alpha-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7-10 mg immunoreactive alpha-glycopolypeptide/6 x 10(8) cells, of which 65-6% was able to associate with native LH beta and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant alpha-subunit was produced as two differently glycosylated forms; an M(r) 23 000 form which contained exclusively N-linked carbohydrate units and another of M(r) 25 000 which appeared to contain additional 0-linked carbohydrate. Data demonstrated that the alpha-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated alpha-subunit is known to be capable of associating with the beta-subunit.

Novel Poly-GalNAcβ1–4GlcNAc (LacdiNAc) and Fucosylated Poly-LacdiNAc N-Glycans from Mammalian Cells Expressing β1,4-N-Acetylgalactosaminyltransferase and α1,3-Fucosyltransferase

“…The recombinant ␣-subunit synthesized by parental CHO Lec8 cells migrated as a broad band with an apparent approximate molecular mass of ϳ25 kDa (Fig. 5), which is similar in mobility to the free ␣-subunit observed for recombinant ␣-subunit expressed in insect cells (25), which have some similarity to Lec8 CHO cells in being unable to terminally glycosylate either N-or O-glycans. By contrast, the recombinant ␣-subunit expressed in L8-GalNAcT cells migrated as a broad band with higher apparent molecular mass extending to ϳ32 kDa (Fig.…”

“…A PCR-amplified DNA fragment starting at bp 72 of the open reading frame of the human ␣-subunit and extending beyond the stop codon was subcloned into the BamHI site of the pcDNA 3.1(ϩ)-TH vector. The resulting vector (pHPC4-␣-subunit) encodes a fusion protein consisting of a signal peptide at the N terminus, followed by an HPC4 linear peptide epitope (23,24), which is followed by the mature peptide of the ␣-subunit (beginning at Ala 25 , the first amino acid after the endogenous, cleavable, signal peptide). The mature HPC4-tagged ␣-subunit protein (after the removal of the signal peptide) has a calculated molecular mass of 11,785.01 Da, but the mature protein contains two N-glycosylation sites and migrates anomalously on reducing SDS-PAGE as a glycoprotein with an apparent molecular mass of ϳ25 kDa (25).…”

“…The resulting vector (pHPC4-␣-subunit) encodes a fusion protein consisting of a signal peptide at the N terminus, followed by an HPC4 linear peptide epitope (23,24), which is followed by the mature peptide of the ␣-subunit (beginning at Ala 25 , the first amino acid after the endogenous, cleavable, signal peptide). The mature HPC4-tagged ␣-subunit protein (after the removal of the signal peptide) has a calculated molecular mass of 11,785.01 Da, but the mature protein contains two N-glycosylation sites and migrates anomalously on reducing SDS-PAGE as a glycoprotein with an apparent molecular mass of ϳ25 kDa (25). CHO Lec8 and L8-GalNAcT cells were transfected with pHPC4-␣-subunit, with or without co-transfection with a plasmid encoding full-length human GalNAc 4-sulfotransferase I (26).…”

High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system

“…The levels of production of rbFSH -and -subunits, in our system, are comparable to those of non-bovine gonadotrophin subunits obtained in the baculovirus system, published elsewhere (Chen et al 1991, Huang et al 1991, Nakhai et al 1991, Jha et al 1992, Sridhar et al 1993, Hasnain et al 1994, Lindau-Shepard et al 1994, Delahaye et al 1996. Levels of production  4.…”

Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus‐infected insect cells

“…Some authors raised the possibility that complex side chains could be produced in insect cells, but followed by rapid removal of terminal and outer sugars by exoglycosidases trimming [19,58]. Sialylation by Sf9 insect cells has only been demonstrated in a few instances [53,59,60], but in a number of cases, sialic acid could be added by Sf9 or Ea4 cells, but removed by neuraminidase activity present in the medium [19,42]. Indeed, several insect cell lines exhibit endogenous neuraminidase activity [61].…”