Serine Protease Inhibitor Superfamily Research Articles

The Major Progesterone-modulated Proteins Secreted into the Sheep Uterus Are Members of the Serpin Superfamily of sErine Protease Inhibitors

The uterine milk proteins (UTMP) are a pair of structurally related basic glycoproteins that when newly synthesized carry phosphorylated mannosyl residues on their carbohydrate chains. They are the major proteins secreted by ovine endometrium under the influence of progesterone. RNA from a late pregnant ewe endometrium was isolated for use in in vitro translation assays and for constructing cDNA libraries. Translation experiments, initially with total cellular RNA and subsequently with RNA selected by hybridization with a specific cDNA, demonstrated the production of two polypeptides (Mr = 47,000 and 55,000) that were precipitated with antiserum to the UTMP. With microsomal membranes in the translation assay, there was increased production of an Mr = 57,000 form that was protected from protease digestion. Antibody screening of a cDNA library in lambda gt11 identified a short clone representing the 3' terminus of the mRNA that was shown by epitope selection experiments to be UTMP specific. This clone was then used to screen a lambda gt10 library. A longer clone (1.3 kilobases) was isolated and sequenced but lacked the 5' terminus to the mRNA. The latter sequence was obtained directly from the mRNA. Interesting features of the UTMP mRNA sequence, which was 1,352 bases long and contained a 1,287-base open reading frame, were two strong start codons, two potential sites for N-glycosylation and a repeat of 21 bases, six bases apart, that resulted in a repeat of seven amino acids. The inferred amino acid sequence agreed closely with the NH2-terminal amino acid sequence obtained directly from the UTMP and clearly placed the UTMP in the serpin superfamily of protease inhibitors. However, we have been unable to demonstrate inhibitory activity toward any serine protease so far tested.

Hormone binding globulins undergo serpin conformational change in inflammation.

A surprising recent finding is that thyroxine binding globulin (TBG) and cortisol binding globulin (CBG), are members of the serine protease inhibitor (serpin) superfamily. Apparently evolution has completely adapted the serpin structure for its new role in these proteins as a transport agent, as there is no evidence of any retained protease inhibitory activity. This drastic change in function raises the question as to why such a complex molecular framework has been selected for the relatively simple task of hormone transport? To function as inhibitors the serpins have a native stressed (S) conformation that makes them vulnerable to proteolytic cleavage, the cleavage being accompanied by an irreversible transition to a stable relaxed (R) form. We demonstrate here that TBG and CBG have retained the stressed native structure typical of the inhibitor members of the family and we provide evidence that the S-R transition has been adapted to allow altered hormone delivery at inflammatory sites.

Site-directed mutagenesis of the reactive center (serine 394) of antithrombin III.

Human antithrombin III (AT) shares significant sequence homology and a common inhibitory mechanism with the serine protease inhibitor (serpin) superfamily. AT has a reactive site in which the P1 residue is primarily responsible for protease specificity. The P1' residue, almost invariably serine, is critical in the inactive natural variant AT-Denver, which has a leucine substitution in that position (Stephens, A.W., Thalley, B.S., and Hirs, C.H.W. (1987) J. Biol. Chem. 262, 1044-1048). In the present study site-directed mutagenesis was used to generate eight variants with altered P1' residues. All were secreted efficiently by COS cells transiently transfected with the AT cDNA in a eukaryotic shuttle vector. All variants also bound heparin as effectively as wild-type AT. Variants were grouped into three categories with respect to thrombin-AT complex formation: 1) no detectable inhibitory activity (proline, methionine); 2) low activity (cysteine, valine, leucine); and 3) near normal activity (glycine, alanine, threonine). The leucine variant, which is in the low activity group, exhibited the same physical and functional properties as AT-Denver. We conclude that the serine hydroxyl is not critical for functional activity and that there is a side chain size optimum which is modulated by hydrophobic effects.

The organization of the human-plasminogen-activator-inhibitor-1 gene. Implications on the evolution of the serine-protease inhibitor family.

Plasminogen activator inhibitor 1 (PAI-1) is a member of the serine protease inhibitor super family (SERPINS) which is thought to play an integral role in the control of plasminogen activation. PAI-1 inhibits both tissue-type plasminogen activator and urokinase-type plasminogen activator and may therefore be implicated in the control of various physiological processes. We have isolated the PAI-1 gene including its 5'-flanking sequence. The gene was characterized by restriction enzyme analysis, Southern blotting and DNA sequencing of all the coding parts as well as the 5'-flanking region. The PAI-1 gene contains nine exons and eight introns distributed over approximately 12.3 kb of DNA. All exon/intron boundaries agree with the 'GT-AG' rule. To characterize the presumptive promoter region, 800 bp of the 5'-flanking region was sequenced and potential binding sites for transacting transcriptional factors were localized. The transcription initiation site was identified by S1 protection experiments and is located 25 base pairs downstream of a TATA consensus sequence. By aligning the gene structure of PAI-1 and four other SERPINS and extrapolating a general tertiary structure to these SERPINS, we find that most introns map between subdomain structures of the proteins. Evidence is presented supporting an intron loss model for the evolution of the SERPIN family.

Organization of the human alpha 2-plasmin inhibitor gene.

We have isolated overlapping phage genomic clones covering an area of 26 kilobases that encodes the human alpha 2-plasmin inhibitor. The alpha 2-plasmin inhibitor gene contains 10 exons and 9 introns distributed over approximately 16 kilobases of DNA. To our knowledge, the number of introns is the highest yet reported for a member of the serine protease inhibitor (serpin) superfamily. All introns are located in the 5'-half of the corresponding mRNA. The 5'-untranslated region and the leader sequence are interrupted by 3 introns totaling approximately equal to 6 kilobases. A "TATA box" sequence is located 17 nucleotides upstream from the proposed transcription initiation site. Multiple "GC box" sequences, G + C-rich sequences, and "CCAAT box"-like sequence, the hepatitis B virus enhancer element-like sequence and the human immunodeficiency virus enhancer-like sequence appear in the 5'-flanking region. The NH2-terminal region, which implements factor XIII-catalyzed cross-linking of alpha 2-plasmin inhibitor to fibrin, is encoded by the 4th exon. The reactive site and plasminogen-binding site, both located in the COOH-terminal region, are encoded by the 10th exon. When similar amino acids of alpha 2-plasmin inhibitor and other members of the serpin gene superfamily are aligned, the position of the 7th intron of the alpha 2-plasmin inhibitor gene aligns precisely with that of the second intron of the genes for rat angiotensinogen and human alpha 1-antitrypsin genes and is misaligned by only one nucleotide with that of the third intron of antithrombin III, suggesting that the alpha 2-plasmin inhibitor gene originates from the common ancestor of these serine protease inhibitors.

Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C 1-inhibitor

Oxidative inactivation of alpha-1-proteinase inhibitor (A1-PI) and plasminogen activator inhibitor-1 (PAI-1), both members of the serine protease inhibitor (serpin) superfamily, using mild oxidation conditions has been already demonstrated. The oxidation mechanism has been shown to involve conversion of methionine to methionine sulfoxide in the reactive center of the inhibitors. In this study evidence is presented that alpha-2-antiplasmin (A2-PI) and antithrombin III (AT III) can also be inactivated by means of oxidation. For total inactivation of 50 pM Al-PI about 10 nM chloramine T (CT) and for the same molar concentration of A2-PI and AT III about 250 nM CT were found necessary. C1-inhibitor (C1-INH) showed some resistance to oxidation that could be overcome only by increasing CT to an amount (> 2000 nM) already beginning to inactivate the corresponding C1-esterase. As target enzymes for A2-PI, AT III, and A1-PI plasmin, thrombin and elastase, respectively, were used. Their activity was not impaired by the oxidation conditions applied. As there is no methionine in the reactive center of AT III an additional mechanism for oxidative inactivation of serpins has to be taken into consideration. Oxidation seems to be a general mechanism for altering the balances between serine proteases and their inhibitors in favour of the protease.

Molecular structure and sequence homology of a gene related to α1-antitrypsin in the human genome

A 7.7-kb EcoRI genomic DNA fragment highly homologous to the human α 1-antitrypsin (AAT) gene has been cloned. This antitrypsin-related sequence is physically linked to the authentic AAT gene and both are present in a single cosmid clone. Nucleotide sequencing of the AAT-related genomic fragment demonstrated extensive homology with the authentic AAT gene in the introns as well as in the exons. The conservation of all RNA splice sites and lack of internal termination codons in the exonic regions suggest that it may not be a classical pseudogene. If expressed, it could result in a protein of 420 amino acid residues exhibiting a 70% overall homology with human α 1-antitrypsin. The signal peptide sequence is well conserved in the related gene, but the active site for protease inhibition of Met-Ser in α 1-antitrypsin has been changed to Trp-Ser. These data suggest that the putative protein encoded by the AAT-related gene is a secretory serine protease inhibitor with an altered substrate specificity. Interestingly, even the intronic regions in the related gene exhibit a 65% overall nucleotide sequence homology with those of the authentic AAT gene. These results suggest that the AAT-related gene is derived from a recent duplication of the authentic AAT gene and represents a new member of the serine protease inhibitor superfamily.

Amino‐acid sequence of human α2‐antiplasmin

The amino‐acid sequence of human α2‐antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature α2‐antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659–1664], 444 residues were identified by amino‐acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369).α2‐Antiplasmin contains two disulfide bridges (Cys64‐Cys104 and Cys31‐Cys113) and four glucosamine‐based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. α2‐Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin).

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily.

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.

Transcriptional regulation by thyroid hormone of an mRNA homologous to a protease inhibitor.

We have previously cloned a cDNA of a rat liver mRNA, designated 4-12B, markedly induced by triiodothyronine (T3) at a pretranslational level [Magnuson, M.A., Dozin, B., & Nikodem, V.M. (1985) J. Biol. Chem. 260, 5906-5912]. Here we show that this hormonal effect is due in part to an increase of the rate of transcription of the 4-12B gene. In addition, the nucleotide sequence of 4-12B cDNA has been determined, revealing significant similarity with the sequences of the superfamily of serine protease inhibitors and a very high homology with contrapsin, a mouse serum trypsin inhibitor, at the level of nucleotide and amino acid sequence (77.9 and 66.8%, respectively). The optimized alignment of the putative reactive center region of 4-12B with four related members of this superfamily revealed that lysine-serine residues are located at the reactive site or adjacent to it, thus suggesting that the triiodothyronine-regulated rat 4-12B mRNA might code for a protease inhibitor with trypsin-like specificity. Although not enough data are presently available to assign definitively antitryptic activity to this protein, the high degree of similarity with members of the superfamily of serine protease inhibitors leaves no doubt that 4-12B is a member of this superfamily.

Isolation and analysis of a cDNA coding for human C1 inhibitor

A cDNA coding for C1 inhibitor was isolated from a human liver λgtll expression library and sequenced by the dideoxy method. The amino acid sequence deduced from the cDNA indicated that the insert was a partial clone coding for 310 amino acids including the reactive site present at the carboxyl end of the molecule. The reactive site corresponds to that previously reported by Salvesen et al . (J. Biol. Chem. 260 , 2432, 1985) . The cDNA also contained a stop codon of TGA, 264 nucleotides at the 3′ noncoding region, and a polyadenylation signal sequence of AATAAA 15 nucleotides upstream from the poly(A) tail. The amino acid sequence flanking the reactive site of the inhibitor is homologous to other members of the superfamily of plasma serine protease inhibitors.

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily.

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.