Influence of glycosylation on the activity of rG-CSFs: in vitro studies: Molecular structure and modeling

R.C. Wade, H. Oschkinat

European Molecular Biology Laboratory Meverhofstrasse 1, Postfach 10.2209 69012 Heidelberg, Germany

Three-dimensional structures of lenograstim have been generated by X-ray [ I ] and nuclear magnetic resonance (NMR) studies. Lenograstim is a member of the 4-helix-bundle family of cytokines which also includes human growth hormone (HuGH), GM-CSF and the interleukins (ILs). Lenograstim comprises 174 amino acids and 4% carbohydrate arranged as four long helices of approximately equal length (A, B. C and D helices) as well as one short helix. Five cysteine residues form two interchain disulphide bonds (cysteine 36-42 and cysteine 64-74), while residue 17 is left free. One O-linked carbohydrate chain is attached to threonine 133. The topology of lenograstim derived from NMR studies is depicted in Fig. 1. Through NMR analysis it is possible, not only to determine the structure of the rHuG-CSF molecule, but also to assess the mobility of different regions of the polypeptide chain as well as the orientation of the sugar moiety in relation to the surface of the protein.
Molecular modelling studies have been performed in an attempt to provide a molecular explanation for the stabilising effects of glycosylation on the rHuG-CSF molecule. A second important consideration is why glycosylation does not seem to affect rHuG-CSF binding with the G-CSF receptor.
Hasegawa proposed that the greater stability of glycosylated rHuG-CSF is due to protection of the only free cysteine residue (position 17) by the sugar moiety. This hypothesis, however, appears unlikely on the basis of structural data. Although the cysteine 17 residue is at the beginning of the A helix, the distance between it and the site of glycosylation (threonine 133) is about 40 A. This distance is too far for a sugar group of about 3 subunits to stretch. Moreover, a sugar group positioned in this way would pass over the B and C helices and, therefore, be expected to influence receptor binding.
There is another possible mechanism. The sugar moiety may confer rigidity to the long flexible C-D loop that contains the threonine 133 residue, making it less susceptible to unfolding or proteolysis. Indeed, preliminary data (V. Gervais, unpublished data) suggest that the C-D loop of glycosylated rG-CSF is not very mobile. These initial studies have produced NMR spectra that indicate a reduction in the flexibility of the amino acid chain at threonine 133 and its neighbouring residues compared with non-glycosylated rG-CSF.
Proteases exhibit a tendency to attack exposed mobile regions of proteins. It is possible, therefore, that through glycosylation, the rHuG-CSF molecule is less vulnerable to proteolytic attack.
Experimental evidence suggests that the G-CSF receptor is a homodimer, although little is known about the mode of G-CSF receptor binding. Modelling and mutational studies with other 4-helix-bundle cytokines (HuGH, GM-CSF, IL-2and IL-4) indicate that the C, A and D helices contact the receptor during binding. However, according to electrostatic analysis of G-CSF receptor binding, the CAD face is not suited to binding a homodimeric receptor because it lacks 2-fold symmetry. In contrast, the BCA face is homogeneous and better suited to binding a homodimer. These results are supported by mutational data which suggest that the B helix is more important for biological activity than the D helix. In conclusion, regardless of which face of the rHuG-CSF molecule is essential for receptor binding, neither the CAD or BCA faces contain the threonine glycosylation site. Glycosylation therefore, is not expected to markedly impair G-CSF receptor binding. However, it is expected that glycosylation has a favourable impact on the stability of the molecule and its resistance to proteolytic degradation.

Intl. J. Hematology (1996) 64 Suppl. 2, S1-S2, S7-S8.

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