No abstract

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed.

No abstract

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 'H NMR signals assignable to three a-linked glucose units at 6 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure aGlc-i-2aGlc__)i3aGlc__<3aMan-+2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the al-6-linked polymannose backbone or addition of al-*3-linked mannose to the core.Glycosylation of asparagine in eukaryotic glycoproteins generally occurs by the transfer of an oligosaccharide with the composition of Glc3Man9GlcNAc2-from the dolichol pyrophosphate oligosaccharide (1). Because of the low concentration of this precursor in cells and the rapid processing of the glycosylated protein, the oligosaccharide is difficult to obtain and its structure was determined by analysis of radiolabeled material isolated from cells incubated individually with radioactive mannose, glucose, N-acetylglucosamine, and galactose. The structure derived by Li et al. (2) is generally accepted, and confirmation has come from other studies (3, 4) and from synthesis of a hexasaccharide with the same presumed partial structure (5).Recently, a new glucosidase-defective mutant of Saccharomyces cerevisiae was obtained that fails to remove the glucose units from the core oligosaccharide after its transfer to protein (6). This mutant makes carboxypeptidase Y that has a slightly reduced mobility on gel electrophoresis and that appears to contain in its oligosaccharide chains all of the glucose present in the lipid-linked precursor. We have now isolated the bulk cell wall mannoprotein from this mutant, recovered the oligosaccharides released by endoglucosaminidase digestion, fractionated them on a sizing column, and analyzed them by proton NMR. The different oligosaccharide fractions show three 1H NMR signals assignable to alinked glucose that are not present in the oligosaccharides from wild-type cells (7,8), and a comparison with the chemical shifts of reference compounds shows that the signals are consistent with the structure assigned to the precursor by Li et al. (2). The NMR spectra also suggest that processing of the core oligosaccharide is otherwise normal because a single al-+2-linked mannose is removed from the aMan-+2 aMan->3aMan >6 side chain, the backbone is extended by addition of mannose in al-*6 linkage, and some al-*3-linked mannose is added to the core. MATERIALS AND METHODSMaterials. The glucosidase-defective strain of S. cerevisiae X2180 (glsI) was originally isolated in combination with the secretion-defective strain sec1...