Isolation and structure determination of the intact sialylated N‐linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

Abstract

Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(Nacetyl-p-glucosaminy1)asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH,. The structures of the carbohydrate chains were determined by 500-or 600-MHz 'H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (lo%), disialylated diantennary (43 YO), disialylated tri-antennary (5%), trisialylated tri-antennary (1 3%), trisialylated tri'-antennary (So/,), and tetrasialylated tetraantennary (1 2%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively a2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing a2-3-as well as a2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an al-6-linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri'-and tetra-antennary oligosaccharide fractions small amounts (< 5%) of compounds were detected having one or more additional Nacetyllactosamine units.Follitropin (follicle-stimulating hormone, FSH) belongs to a heterodimeric glycoprotein hormone family in which each hormone is composed of two non-covalently linked subunits, denoted a and / 3. The amino acid sequences of the a subunits are essentially identical, whereas those of the fl subunits are hormone-specific. The structures of the carbohydrate chains differ between the glycoprotein hormones and are believed to confer hormone specific-functions [l -31.FSH is synthesized by the gonadotropes in the anterior pituitary and is required for normal reproductive functioning in both females and males. There is a need of FSH for clinical purposes since it is applied, either alone or in combination with human LH, for controlled ovarian hyperstimulation in in vitro fertilization [4,5]. Human FSH is also used to stimulate the development of ovarian follicles in anovulatory women suffering from luteal phase deficiency [6], or chronic anovulatory syndrome [7].Correspondence to J. F. G. Vliegenthart, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, Transitorium 111, P. 0. Box 80.075, NL-3508 TB Utrecht, The NetherlandsAbbreviations. PNGase-F, peptide-N4-(N-acetyl-P-glucosaminy1)asparagine amidase F ; NeuSAc, N-acetylneuraminic acid; Fuc, Lfucose; hFSH, human follicle-stimulating hormone (follitropin); LH, luteinizing hormone (lutropin); hCG, human chorionic gonadotropin; CHO, Chinese hamster ovary; t-PA, tissue plasminogen activator.Enzymes. Peptide-N4-(N-acetyl-~-glucosaminyl)asparagine a...

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

“…The presence of two sialic acid residues in both structures is proven by the relative intensities of the NAc, H3a, and H-3e structural reporters of NeuSAc, as compared to the 'H-NMR spectrum of N3.2. The structural-reporter-group signals of the three antennae in N2.4B match those of the non-fucosylated analogue N2.3 in [44]. The tri'-antennary character of N2.4A is demonstrated by the H-1 signals of Man4 and Man-4' at 6 5.125 and 6 4.868, respectively, the H-2 signals of Man-3, Man4 and Man-4' at 6 4.250,6 4.196, and 6 4.090, respectively, and the H-6 signal of M a n 4 at 6 4.210.…”

“…3). Fractions N1, N2, N3, and N4 have the same retention volumes as reference monosialylated diantennary, disialylated diantennary, trisialylated tri-/tri'-antennary, and tetrasialylated tetraantennary oligosaccharides, respectively [44]. The remaining peaks eluting before N1 and after N4 do not correspond with carbohydrate material (resorcinolhlfuric acid asay) .…”

“…compound N4.2B in [44]; see Table 4), and that of N4.3 a modification of N4.2, in which one of the NeuSAc residues has been replaced by a NeuSGc residue (cf. compound A4.5 in [24]; see Table 4).…”

“…compound N3.2A in [44]; see Table 4); and a major component (75 %) : fucosylated, a(2-3)-trisialylated (Neu5Ac) ti'-antennary oligosaccharide N3.2A (cf. compound N3.3 in [44] ; see Table 4).…”

“…compound N3.2A in [44]; see Table 4); and a major component (75 %) : fucosylated, a(2-3)-trisialylated (Neu5Ac) ti'-antennary oligosaccharide N3.2A (cf. compound N3.3 in [44] ; see Table 4). The position of Man-4' H-2 at 6 4.090 was recently established by 2D-ROESY, and turned out to be specific for tri '-[46] and tetraantennary structures (unpublished results).…”