Isolation and Properties of Recombinant DNA Produced Variants of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 7, Issue of April 10,pp. 4384-4389 1985 Printed in d.S.A.

Isolation and Properties of Recombinant DNA Produced Variants of Human al-Proteinase Inhibitor* (Received for publication, October 3, 1984)

James Travis$$, Maurice Owenfl,Peter Georgell, Robin Carrellll,Steven Rosenbergll, RobertA. HallewellII , and Philip J. Barr11 From the $Departmentof Biochemistry, University of Georgia, Athens, Georgia 30602, the TDepartment of Pathology, Christchurch Hospital, Christchurch, New Zealand, andthe 11 Chiron Corporation, Emeryville, California 94608

Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human al-proteinase inhibitor, representing 10%of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated withone being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, andbothhad shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasmaal-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing proteinwas readily inactivated. The specificity of alproteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or pancreatic elastase, “trypsin-like” serineproteinases not being inactivated at all. These data indicate the potential importance of mutant forms of proteinase inhibitors, produced by recombinantDNA technology, as therapeutic agents for the inactivation of excess proteinases of a specific type in tissues.

induced deficiency (4) or by oxidative inactivation due to conversion of the reactive site methionine to its sulfoxide derivative ( 5 ) ,enzymatic degradationof lung connective tissue may occur and, ultimately, pulmonary emphysema(6). In order to decrease the proteolytic burden on the lung, attempts have been made to either synthesize effective low molecular weight inhibitors for therapeutic use (7, 8) or to develop techniques for the isolation of large amounts of the naturalinhibitor for supplementation (9). Recently (IO), cDNA clones of normal al-PI have been constructed in plasmids and the protein expressed, thereby providing a potential source for the isolation of large quantities of inhibitor. However, such a protein would still be susceptible to oxidative inactivation, thereby reducing its potential effectiveness. Because neutrophil elastasecleaves peptide bondspreferentially after valyl residues (2), a mutated form of al-PI with valine in the Pl reactive site position (11)would seem a good potential choice as an inhibitorwith therapeutic value. Previously (lo), it had beenshown that sucha protein could be produced in yeast by mutagenesis of the cDNA sequence codingfor the reactive site sequence. Furthermore, extractsof yeast expressing this protein inactivated neutrophil elastase both before and after oxidationby N-chlorosuccinimide, while those containing the normal al-PI sequence lost all activity after this treatment. In this report we describe the isolation and properties of both the normal (al-PI yeast methionine) and the The abnormal degradation of the connective tissue proteins mutated form (al-PIyeast valine) of this inhibitor. of the lung is believed t o be due to uncontrolled proteolysis EXPERIMENTALPROCEDURES by an elastolytic enzyme released from neutrophils (1).This enzyme degrades elastin, collagen, and proteoglycan (2) and, Materials unless controlled, could cause extensive lung damage. NorHuman neutrophil elastase (12) and human neutrophil cathepsin mally, the plasma protein al-proteinase inhibitor (al-antiG (13)were obtained aspreviously shown. Porcine pancreatic trypsin trypsin) (al-PI1) rapidly inactivates neutrophil elastase (3), and porcine pancreatic elastase were from Sigma. Human plasmin thereby reducing tissue proteolysis. However, when thelevels and human Factor Xa were gifts of Dr. John Fenton andDr. Robert of active inhibitor arereduced, either because of a genetically Jordan, respectively. Normal humanal-PI and the Pittsburgh mutant form of al-PI were isolated as described elsewhere (14, 15). The S and Z variants of a,-PI were prepared by the method of Jeppsson et al. (16). Rabbit anti-human al-PI was obtained from Behringwerke. Restriction enzymes and theKlenow fragment of DNA polymerase I were obtained from New England Biolabs. The neutrophilelastasesubstrate t-Boc-L-Ala-L-Ala-L-NorvalSBz, the cathespin G substrate t-Boc-L-Ala-L-Ala-L-Phe-Sbz, and the plasmin substratet-Boc-L-Ala-L-Ala-L-Arg-SBz were gifts of Dr. James C. Powers. Bz-Ile-Glu-(&OR)-Gly-Arg-pNA was from Kabi. N-Suc-L-Ala-L-Ala-L-Ala-pNA and Bz-L-Arg-OEt were from Sigma.

* This research was supported in partby grants from the National Institutes of Health, TheCouncil for Tobacco Research-U. S. A,, and the Medical Research Council and the National Children’s Health Research Foundation of New Zealand. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J To whom correspondence should be addressed. The abbreviations used are: al-PI, al-proteinase inhibitor;t-BocL-Ala-L-Ala-L-norval-SBz, t-Boc-L-alanyl-L-alanyl-L-norvalyl-thioMethods benzyl ester; t-Boc-L-Ala-L-Ala-L-Phe-SBz, t-Boc-L-alanyl-L-alanylConstruction of Yeast Expression Plasmids-DNA manipulations L-phenylalanyl-thiohenzyl ester; t-Boc-L-Ala-L-Ala-L-Arg-SBz; tBoc-L-alanyl-L-alanyl-L-arginyl-thiobenzyl ester; Bz-Ile-Glu-(&OR)- were carried outas previously described (10,17). Plasmid PAT (Met), Gly-Arg-pNA, benzoyl-isoleucyl-glutamyl-ornithyl-glycyl-arginylpar-containing an al-PI cDNA extending from Asp-2 to the C terminus anitroanilide; Bz-L-Arg-OEt, benzoyl-L-arginylethyl ester;-Sue, suc- (IO),was digested with BamHI, made flush-ended using the Klenow fragment of DNA polymerase I , digested with Sal1 and the 1200-bp cinyl-; NaDodSOI, sodium dodecyl sulfate; PMSF, phenylmethaneal-PI fragment isolated from a 1%agarose gel. sulfonyl fluoride; bp, base pair; kh, kilobase pair. 4384

al-Proteinase In~ibitorV a r ~ ~ t s PlasmidpPGAP, whose properties will be described indetail elsewhere: is a derivative of pBR322 (18) and was prepared as follows. pBR322 was digested with EcoRI and SalI, these sites converted to BamHI sites by the use of asynthetic DNA linker (5'GGATCCGGATCC), and the molecule then ligated to produce a deleted derivative (about 3.8 kb) with a single BamHI site. A 1300bp BarnHI fragment containing the yeast glyceraldehyde 3-phosphate 49 gene (19) promoter and transcription terminator was inserted at this BamHI site. This fragment consists of about 400 bp corresponding to the 5' untranslated region of the glyceraldehyde-3-phosphate dehydrogenase and contains the promoter as well as 900 bp corresponding to the entire 3' untranslated region extending from the natural SalI site which is about 60 bp from the glyceraldehyde-3phosphate dehydrogenase translation termination codon tothe BamHl site. These two fragments were linked using a 35-bp synthetic DNA fragment c o n ~ i n i n gan Ne01 site (B'CCATGG) at the gtyceraldehyde-3-phosphate dehydrogenase initiator methioninea t one end and a Sat1 site at the otherfor joining to thenatural Sal1 site of the glyceraldehyde-3-phosphate dehydrogenase gene as described above. Plasmid pPGAP was prepared as shown in Fig. 1 and used to clone the 1200-bp al-PI coding sequence described above. The resulting plasmid was partially digested with RanHI, and a 2.5-kb fragment containing the (ul-PI cDNA flanked by the yeast glyceraldehyde-3phosphate dehydrogenase promoter and transcription terminator was isolated. This fragment was inserted into the yeast shuttle vector pC1/1 (10,20) a t its BarnHI site and a recombinant clone containing the 2.5-kb fragment in the orientation shown was obtained. An analogous plasmid to pCl/lGAPal-PI with Met-358 changed to Val-358 was prepared as follows. A 1030-bp BamHI-BstE2 fragment containingthe glyceraldehyde-3-phosphate dehydrogenase promoter and N-terminal half of the nl-PI cDNA was isolated from plasmid p C l / l G A P ~ ~ - PA~BstE2-BamHI . fragment of 1440bp, containing the C-terminal half of the nl-PI cDNA and the gfyceraldehyde-3-phosphate dehydrogenase terminator where the Met-358 to Val-358 mutation had been engineered by site-specific mutagenesis, was isolated from pCl/lPH05AT (Val) (10). These fragments were ligated with the pC1/1 vector which had been treated with BamHI and alkaline phosphatase, and a plasmid with the same structure as pCl/lGAP al-PI with the Met-358 to Val-358 substitution was obtained. Yeast Transformation and Growth-Yeast strain AB110 (Mat), urd-52, leul-04 or leu2-03 and leu2-112,pep4-3, his4-580, [cir"] was transformed with plasmids pC1/lGAPcul-PI(Met) and pC1/lGAPaIPI(Val) and leucine prototrophs were selected (10, 21). Transformants were grown in 2-ml cultures using synthetic complete medium lacking leucine (22). Large scale cultures were grownin YEP medium (23) to an Amam of10-12. Yeast was harvested by centrifugation, washed once with distilled water, and stored a t -80 "C. Isolation of Yeast-derived al-Proteinase Inhibitor Variants-Preliminary experiments had indicated that extracts containing q - P I rapidly lost inhibitory activity, presumably due to the presence of yeast proteinases. Therefore, extraction buffers utilized in the purification of al-PI routinely contained 1 mM EDTA and 1 mM PMSF. Both inhibitors were purified by the procedures described below and behaved identically during the isolation. Extraction-Typically, 14 g (wet weight) of yeast containing the