Mutations affecting the activity of urokinase-type plasminogen activator.

Mutagenesis throughout the single-chain urokinase-type plasminogen activator (scu-PA) cDNA molecule, followed by expression of the mutant genes and secretion of the resulting mutant proteins from yeast, has been used to determine the amino acid residues important for activity of scu-PA molecules. Twelve out of 13 colonies secreting variant scu-PA molecules with decreased ability to form a zone of fibrinolysis had mutant genes with a single codon alteration in the serine protease encoding domain (B-chain). Many of these changes are of highly conserved residues in the serine proteases and are consequently of considerable interest. A model three-dimensional structure of the protease domain of urokinase was used to explain the basis for the effects of these down mutations. The model showed that the strongest down mutations result from either interference of the mutated side chain with substrate binding at the active site or the introduction of bulky or charged groups at structurally sensitive internal positions in the molecule. Attempts to find second site revertants of five down mutants, altered either at the plasmin activation site or near the serine at the active site, only resulted in same-site revertants, with the original or closely related amino acids restored.

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.

Glycosylation and secretion of human alpha-1-antitrypsin by yeast.

Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver. To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter. Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase. All carbohydrate is removed by endoglycosidase H treatment. Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.

Nucleotide sequence of the RAD10 gene of Saccharomyces cerevisiae.

The RAD10 gene is one of several genes in Saccharomyces cerevisiae required for incision of u.v.-irradiated or cross-linked DNA. We have determined the nucleotide sequence of the RAD10 gene and its flanking regions. The RAD10 nucleotide sequence presented here differs significantly from that recently reported. The RAD10 protein predicted from the nucleotide sequence contains 210 amino acids with a calculated mol. wt. of 24 310. The middle portion of the RAD10 protein, which is highly basic and also contains eight of the total of 10 tyrosine residues present in the protein, may be involved in DNA binding by ionic interactions and tyrosine intercalation between the bases of DNA. A genomic deletion of the entire RAD10 gene does not affect viability; however, the rad10 deletion mutant is highly u.v. sensitive.

Molecular cloning of the RAD10 gene of Saccharomyces cerevisiae.

We have cloned the RAD10 gene of Saccharomyces cerevisiae and physically mapped it to a 1.0-kb DNA fragment. Strains containing disruptions of the RAD10 gene were found to show enhanced UV sensitivity compared with the previously characterized rad10-1 or rad10-2 mutants. The UV sensitivity of the disruption mutant is comparable to the highly UV sensitive rad1-19, rad2-delta, and rad3-2 mutants.