Characterization of the Glycosylation on Recombinant Human Low-Affinity Nerve Growth Factor Receptor

Abstract

To study the structure of the nerve growth factor (NGF) binding region in this glycoprotein, a recombinant form (rhNGF-R) was prepared that contains the N-terminal 222 amino acids ending just before the transmembrane region of the native full length receptor. rhNGF-R is a soluble form of the receptor that retains its ability to bind NGF. This protein contains one consensus site for N-linked glycosylation at Asn-32 and a Ser, Thr, and Pro-rich C-terminal domain that may contain O-linked glycosylation. Enzymatic deglycosylation combined with reductive amination derivatization strategies previously developed in this laboratory was used to determine the structure and branching of the majors-linked carbohydrates on rhNGF-R. Carbohydrate analysis using high pH anion exchange chromatography with pulsed amperometric detection and enzymatic deglycosylation, combined with reductive animation derivatization strategies, was used to determine the structure and branching of the major N-linked carbohydrates on rhNGF-R. The structures were determined to be fucosylated diantennary complex oligosaccharides.