Simple method for large-scale production of macrophage activating factor GcMAF

Yoko Nabeshima,Takeshi Kawauchi,Chiaki Abe,Yo-Ichi Nabeshima,Tomoko Hiroi,Yoshihiro Uto

doi:10.1038/s41598-020-75571-y

Abstract

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Highlights

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans
The protein in the de-glycosylated fraction (Fig. 2B (+))was detected with Helix promatia (HPA) lectin, suggesting that Gc1FHis protein bearing O-linked trisaccharide (i.e., Sialic acid-Galactose-GalNAc-Thr420)[20,21,22,23] was synthesized in CHO cells, and that sialic acid and galactose were removed by the de-glycosylation treatment (Fig. 2B (+))
This result is consistent with reports that Gc protein prepared from human blood can be converted to HPA lectinreactive GcMAF by treatment with β-d-galactosidase and sialidase[34,35,36]

Summary

Introduction

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Current detailed glycan structural analyses using glycosidase treatment and mass spectrometry[6,20,21,22] indicated that (1) Gc1F and Gc1S proteins have the same linear trisaccharide, sialic acid-galactose-GalNAc, on the T hr[420] residue (Fig. 1 Model B)[23] and (2) substitution of a lysine residue at the position corresponding to T hr[420] in Gc2 prevents this isoform from being glycosylated at that position, and Gc2 is not glycosylated (Fig. 1) These conclusions are supported by the finding that Gc1 from cancer patients contains the same trisaccharide as Gc1 from healthy volunteers, namely, sialic acid-galactose-GalNAc-Thr42024. There is a need for methodology to provide a consistent product on a large scale for further studies

Methods

Results

Conclusion