Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast.

Using site-directed mutagenesis, we have changed the asparagine in human single-chain urinary plasminogen activator (u-PA) at position 302 to an alanine. This alteration removes the only known amino acid residue glycosylated in the protein. The single-chain u-PA containing an alanine residue at position 302 instead of asparagine (scu-PA(N302A] cDNA gene was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein product into the culture broth was achieved by replacing the human secretion signal codons with those from yeast invertase, adding a yeast promoter from the constitutively expressed glycolytic genes triosephosphate isomerase or phosphoglycerate kinase, and integrating multiple copies of these transcriptional units into the genome of yeast strains carrying the "supersecreting" mutation ssc1. When fermented in a fed-batch mode, these recombinant baker's yeast strains secreted scu-PA(N302A) in a strongly growth-associated manner. Greater than 90% of the u-PA found in the culture broth was in the single-chain form. Scu-PA(N302A) was purified to homogeneity using two chromatography steps. The purified protein had a molecular weight of 47,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lacked any detectable N-linked glycosylation. The in vitro fibrinolytic properties of scu-PA(N302A) were found to be essentially equivalent to those of natural single-chain u-PA derived from the human kidney cell line TCL-598. Since scu-PA(N302A) lacks the immunogenic N-linked carbohydrate pattern of yeast, it may be a useful therapeutic agent which can be produced economically by yeast fermentation.

Properties of the nerve growth factor receptor. Relationship between receptor structure and affinity.

Microsomal membranes from A875 human melanoma cells contain nerve growth factor receptors (NGF-receptors) which appear to belong to a single class with homogeneous binding properties, as determined by Scatchard plots. NGF-receptors in these membrane preparations are also uniformly highly sensitive to tryptic proteolysis, and 125I-NGF bound to NGF-receptor in these membranes is rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, analysis of 125I-NGF dissociation kinetics indicated that two classes of NGF-receptor were present in these membranes. Thus, NGF-receptors can express either high or low affinity trypsin-sensitive states in addition to the high affinity trypsin resistant NGF-receptor state described previously (Buxser, S. E., Kelleher, D. J., Watson, L., Puma, P., and Johnson, G. L. (1983) J. Biol. Chem. 258, 3741-3749). The high affinity trypsin-sensitive and low affinity trypsin-sensitive states correlate with 200- and 90-kDa 125I-NGF X NGF-receptor complexes observed in photoaffinity cross-linking experiments. The absence of differences in peptide maps generated from the two sizes of NGF-receptor proteins together with structural and binding data strongly indicates that the 200-kDa NGF-receptor protein is a complex, probably a dimer, consisting of two 80-kDa NGF-receptor proteins associated with a single beta-NGF dimeric molecule. A model is proposed which relates structural states of NGF-receptors with specific receptor binding properties. The model provides an alternative explanation for binding phenomena previously attributed to negative cooperativity.

Change in state of nerve growth factor receptor. Modulation of receptor affinity by wheat germ agglutinin.

The binding of 125I-labeled nerve growth factor (NGF) to human melanoma cell (A875) membranes, detergent-soluble membrane extracts, and membrane extracts reconstituted into phospholipid vesicles was significantly increased when binding was carried out in the presence of wheat germ agglutinin (WGA). In the absence of WGA, all 125I-NGF binding was rapidly eliminated by trypsin treatment or rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, in the presence of WGA, up to 75% of 125I-NGF bound was resistant to trypsin digestion and was only slowly dissociated by a high concentration of unlabeled NGF. The effects of WGA can be blocked or reversed by N-acetylglucosamine. Both WGA and NGF rapidly associate with soluble extracts and reconstituted vesicles and, at the concentrations used here, reach binding equilibrium within 2 min. The conversion to slowly dissociating, trypsin-resistant binding, however, was not complete for at least 10 min. Both WGA and NGF are required for maximum accumulation of trypsin-resistant, slowly dissociating binding. The order of addition of NGF and WGA has no effect on the rate of conversion of NGF-receptor, and the conversion occurs after both NGF and WGA are present. The amount of conversion is dependent on the incubation temperature, and significantly greater conversion occurs at 37 than at 0 degrees C. The generation of the trypsin-resistant, slowly dissociating state of NGF-receptor is consistent with a time- and temperature-dependent conformational change in NGF-receptor which occurs after interaction of both NGF and WGA with the receptor or closely associated structures.

Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography.

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.