Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells

Abstract

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.

Introduction

Follistatin (FS), a glycoprotein, was first discovered in ovarian follicular fluid as an inhibitor of pituitary follicle-stimulating hormone secretion [1], [2]. Subsequent studies have revealed that FS can bind to activins and neutralize their biological activities [3], [4]. Activins are members of the transforming growth factor-β superfamily, and they play important roles in the regulation of cell growth and in the differentiation processes that lead to morphogenesis in early vertebrate development [5], [6]. Since FS and activins are broadly distributed, they are not confined solely to tissues associated with reproduction [7].

FS is present in heterogeneous forms [8]. The FS gene consists of 315 amino acids, and it includes six exons (Fig. 1); alternative splicing can generate two isoforms, i.e. a 315-amino-acid protein (the full-length form, FS315) and a 288-amino-acid protein (the carboxy-truncated form, FS288) [9]. The activin-neutralizing activity of FS288 is higher than that of FS315 [10], [11], which appears to correlate with their heparin/heparan sulfate proteoglycan-binding abilities [12]. The heterogeneity of FS is also due to diverse glycosylation. FS has two potential N-glycosylation sites (Asn95 and Asn259). Oligosaccharides are generally known to play important roles in defining the properties of glycoproteins such as their biological activity, immunogenicity, pharmacokinetics, solubility, and protease resistance [13], [14]. Glycosylation on FS is also likely to exert an effect on activin-neutralizing activity; however, neither structure of the N-linked oligosaccharides in FS, nor their physiological roles, have been clarified due to the limited availability of these oligosaccharides.

The aim of this study was to elucidate the glycosylation of FS. We previously developed an oligosaccharide profiling method using liquid chromatography/mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) [15], [16], [17], [18], [19], [20], [21], [22]. Recently, we demonstrated a procedure for facilitating the structural analysis of glycoproteins [16]. Carbohydrate profiles and site-specific glycosylations can be characterized by the GCC-LC/MS method, followed by mass spectrometric peptide/glycopeptide mapping. We used this method to demonstrate here the carbohydrate heterogeneity and the site-specific N-linked oligosaccharide structures in recombinant human FS288 (rhFS) produced in Chinese hamster ovary (CHO) cells, in which a sufficient amount of FS could be expressed.