Siiyama (serine 53 (TCC) to phenylalanine 53 (TTC)). A new alpha 1-antitrypsin-deficient variant with mutation on a predicted conserved residue of the serpin backbone.

alpha 1-antitrypsin (alpha 1AT), a plasma serine protease inhibitor, increases the risk of precocious pulmonary emphysema in individuals when deficient. Although more than 25 years have passed since a deficiency in the serum level of alpha 1AT was reported, it is only recently that the consequence of the amino acid replacement which leads to the deficient state has been discussed in terms of the crystallographic structure of alpha 1AT and the amino acid residues conserved in the superfamily to which it belongs. Our case involved a 38-year-old Japanese male with alpha 1AT deficiency which was analyzed and identified as a new deficient variant. The serum alpha 1AT of the proband migrated to the S position of the reference serum which is more cathodal than M1, the predominant normal variant, when isoelectric focusing (pH 4.2-4.9) is performed by a combination of Western blotting and crossed immunoelectrophoresis. The new deficient variant is designated as Siiyama after his birthplace. Although liver biopsy specimen showed no apparent pathological findings, PAS-positive with diastase-resistant inclusion bodies and immunoreactive aggregates were detected in several hepatocytes. In addition, similar alpha 1AT mRNA transcript levels were observed in peripheral blood leukocytes from the proband and healthy subjects by Northern analysis. All the coding exons (exon Ic, II, III, IV, and V) of the alpha 1AT gene of the proband and his family were amplified by polymerase chain reaction and followed by direct sequencing. A single missense mutation, Ser53 (TCC) to Phe53 (TTC was identified in exon II of the proband's alpha 1AT gene. All his family examined were heterozygous at this base. Ser53 is one of the most conserved residues as predicted by Huber and Carrell (Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966) and is thought to contribute to the organization of the internal core element of the alpha 1AT molecule. The mutational matrix number of Ser to Phe substitution is -3, indicating that this change is evolutionally rare. In this regard, a possible explanation for the deficient state in alpha 1AT Siiyama is that the change from an uncharged polar to a nonpolar amino acid imposed on the conserved serpin backbone exerts severe effects on the integrity of the molecule, and hence alters the intracellular processing of alpha 1AT.

I To whom correspondence and reprint requests should be addressed Dept. of Respiratory Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. exerts severe effects on the integrity of the molecule, and hence alters the intracellular processing of alAT.
It is well recognized that reduced serum levels of al-antitrypsin (a1AT)' increase the risk of early-onset pulmonary emphysema in individuals with this deficiency (2-5). alAT is highly pleomorphic and plays a physiological role in the inhibition of neutrophil elastase, a powerful serine protease (2)(3)(4)(5). Crystallographic analysis of the molecule has provided an understanding of structure and function relationships in alAT and other proteins belonging to the serpin superfamily (1, 6, 7). Among these, alAT has been extensively studied and researched because of its association with human disorders. alAT is coded by a single-copy gene spread over 12.2 kilobases on chromosome 14q 31 to 32.3 (3, 8, 9). Serum alAT, a 52-kDa glycoprotein, is thought to diffuse into the alveolar space and irreversibly inhibit neutrophil elastase that digests elastin and most tissue matrix proteins (3, 4, 10). In the normal alveolar space with sufficient a1AT molecules, the effects of neutrophil elastase are neutralized, and the lower respiratory tract is protected from proteolytic attack. In the a1AT-deficient state, the affected individuals are faced with the risk of developing pulmonary emphysema, usually between the age of 30 to 40 and with occasional liver damage (3,4,10,11).
More than 30 alAT genes of both normal variants and abnormal variants with clinical consequences have been analyzed at the level of nucleotide alteration (2-4). Among them are 11 clinical variants with amino acid substitutions. Utilizing an interpretation of the crystal structure of alAT, Huber and Carrell (1) recently reported on critical amino acid residues which are conserved in the serpin superfamily. When they examined natural serpin variants including alAT, antithrombin 111, C1-inhibitor, heparin cofactor 11, and antiplasmin, six clinically significant alAT variants were found at predicted conserved residues or on residues close to the predicted locations.
While the gene frequency for the Z-type a1AT variant is relatively high (0.01-0.02) among Caucasians (2-4), deficient variants among Orientals are rare, with only one deficient variant elucidated at the gene level (12). In this study, we analyzed a new alAT deficient case in Japan and found that the mutation occurs at one of the residues that Huber and Carrell (1) regard as conserved and predict the effect of the substitution in the crystalline structure.

MATERIALS AND METHODS
Study Group-A 38-year-old male was admitted to the Hokushin General Hospital because of exertional dyspnea. Chest x-ray films and CT scans showed overinflated lungs and bullous changes especially in the lower lung fields. Obstructive ventilatory impairment (FEV,,%: 31.5%, FEVl.o: 1.73L) was observed. The serum level of n l A T was 14.5 mg/dl (normal range 148 -317 mg/dl). Laboratory data showed no apparent liver dysfunction. The family members of the proband available for evaluation included his parents, elder sister, and daughter (Fig. 1). Their n1AT levels in serum were 106 mg/dl (father, 111-3), 119 mg/dl (mother, III-14), 87 mg/dl (sister, IV-2), and 140 mg/dl (daughter, V-4), respectively, all showing about half the normal level. Consanguinity was recorded in the patient's history; his parents are cousins (Fig. 1).
Western blotting was performed after IEF. The proteins were transferred to Immobilone (polyvinylidene difluoride, 0.45 pm, Millipore) using a buffer containing 25 mM Tris, 192 mM glycine, 20% methanol, and 0.7% acetic acid at constant voltage of 15 V for 12 h at 4 "C. The membrane was incubated with Block Ace'" (Dainihon Seiyaku Co. Ltd, Japan) for 1 h. After rinsing three times for 10 min, it was incubated with 1/500 diluted anti-human a1AT antibody (Dako) for 1 h. After rinsing three times for 10 min, the membrane was incubated with 1/1000 diluted horseradish peroxidase-conjugated protein A (Bio-Rad) for 1 h. After rinsing three times, the membrane was stained in 50 mM Tris-HCI, p H 7.4, containing 0.40 mg/ml 3.3'diaminobenzidine tetrahydrochloride and 0.04% H202. Phosphatebuffered saline containing 0.05% Tween 20 was used as a buffer for dilution and rinsing of antibody and horseradish peroxidase-conjugated protein A. All procedures were performed at room temperature.
Crossed immunoelectrophoresis was performed as follows. IEF gel strip (70 X 4 mm) was cut and placed in 1% agarose gel (agarose A, Pharmacia) in a barbital buffer (pH 8.6, ionic strength 0.05) with 1/ 480 -960 diluted anti-human a l A T antibody. Constant voltage of 50 V for 6 h was applied to the gel with barbital buffer as the electrode buffer. After completion of the procedure, the gel was rinsed with phosphate-buffered saline for 30 min, three times, then immersed in 1/100 diluted horseradish peroxidase-conjugated protein A for 1 h. After rinsing three times with PBS for 30 min, the gel was stained in 50 mM Tris-HC1, pH 7.4, containing 0.40 mg/ml 3,3'-diaminobenzidine tetrahydrochloride and 0.04% H202.
Immunohistochemical Staining of the Liuer-Formalin-fixed, par- Serum nlAT levels (normal range 148 -317 mg/dl) are shown below the symbols (0, males; 0, females). The inheritance of the a1AT Siivnmn gene is shown as black in the individuals included in this study. There was a consanguineous marriage recorded in the patient's history; his parents are cousins.
affin-embedded liver tissues were cut into 3-pm thick sections, deparaffinized, and rehydrated. Immunoperoxidase staining was performed with an avidin-biotin complex as described by Hsu et al. (13).
Polymerase Chain Reaction (PCR) and Sequencing of a l A T Gene-Genomic DNA was obtained from peripheral blood leukocytes according to the method described by Jeffreys and Flavell (14). Synthetic oligonucleotide primers were prepared so as to cover all the coding exons of the a1 AT gene (Applied Biosystems DNA synthesizer 341A). Each exon of the nlAT gene was amplified separately by PCR with Taq polymerase (Perkin-Elmer-Cetus) using a Thermal cycler (Perkin-Elmer-Cetus) under the recommended conditions of the supplier. Amplification was performed with 30 cycles of denaturation (94 "C, 1 min), annealing (55 "C, 1 min), and extension (72 "C, 2.0 min). Amplified products were electrophoresed in 2% low melting agarose gel (Seaplaque'", FMC BioProducts), enucleated and extracted with phenol/chloroform, and precipitated with ethanol. The purified DNA was used as a template in the next PCR with an asymmetrical primer ratio (50 pmol uersw 1 pmol) which was performed for 25 cycles of the same thermal cycle setting to generate single-stranded DNA suitable for sequencing. Sequencing was carried out in both directions with Sequenase" (United States Biochemical) as instructed by the supplier.
RNA Analysis of Peripheral Blood Leukocytes-alAT mRNA transcripts of leukocytes were evaluated by Northern analysis. Leukocytes m4;Ml

FIG. 2. Western blotting and crossed immunoelectrophoresis analysis of serum alAT Siiyama after IEF. A, Western
blotting of alAT. After conventional IEF, the proteins were transferred to Immobilon" (see "Materials and Methods"). For the reference sera a1AT MlS, MlX, and MlY,,,,,,, 2.5 p1 of one-seventhdiluted serum was loaded on IEF. For a l A T ZZ 2.5 p1 of one-halfdiluted serum, and for the proband 2.5 pl of undiluted serum wereused, respectively. The anode is at the top and the cathode is at the bottom. Lane 1, a l A T M1S; lane 2, a l A T M 1 X lane 3, a1AT MIYlomnlo; lane 4, a l A T ZZ; lane 5, proband. Note that in lane 5, three faint bands (arrowhead 1-3) could be observed, indicating the possibility of either phenotype S or Z. B, crossed immunoelectrophoresis of a1AT. The polyacrylamide gel after IEF was enucleated and placed in the vertical direction (anode at the top and cathode at the bottom in the first IEF). For reference serum of MlZ, 2.5 p1 of one-seventh-diluted serum was loaded on IEF, and for ZZ and the proband the amounts loaded on IEF were the same as described in the legend for Fig. 2A.
The immunoelectrophoresis was performed in the horizontal direction (anode at the right and cathode at the left). Panels 1 and 2, proband; panel 3, a1AT ZZ; panel 4, a1AT M1Z. In the case of lane I , agarose gel containing 1/96O-diluted anti-human a l A T antibody was used. In the case of lanes 2-4, agarose gel containing 1/480-diluted antibody was used. Note in lanes 1 and 2 two broad precipitation peaks could be observed at the position corresponding to S but not to Z. were collected from blood by the dextran method (15), and their total RNA was obtained using RNAZol" (CinnaDiotecx Laboratories). Northern analysis was performed with 1% agarose gel electrophoresis under denaturing condition, blotted to NYTRAN" (Schleicher & Schuell), and hybridized with a 3"-labeled probe. The probe used was prepared from genomic DNA of a normal subject by the amplification of a part of exon I1 (nucleotide numbers 5464 -5979) of the tvlAT gene (8) and labeled with a random primed DNA labeling kit (Boehringer Mannheim). Exposure of the autoradiograms was 4 days at -70 "C.

Determination of the Phenotype of a New alAT-deficient
Variant on IEF-In contrast to the Z type-deficient alAT, banding of alAT of the proband could not be observed by conventional IEF using Coomassie Brilliant Blue R250 as the stain, but by a combination of IEF and crossed immunoelectrophoresis followed by immunoperoxidase staining the IEF phenotype of the proband serum was determined as the alAT S-type (Fig. 2). When Western blotting was performed after IEF, the proband serum showed three faint bands, indicating the possibility of the S or Z type with the reference sera MlS, MlX, MIYto,,t,, and ZZ (lane 5, Fig. 2 A ) . Crossed immunoelectrophoresis using the cut-out gel after IEF revealed two peaks which correspond to the major 4 and major 6 bands of S a1AT (panel 1, Fig. 2B; also see lanes 1, 4, and 5 of Fig.  2 A ) . We designated this deficient phenotype as Siiyams after the proband's birthplace in Japan.

Histological Findings in Biopsied Liver Specimen-AI-
though no apparent pathological findings were observed after routine hematoxylin-eosin and silver staining (not shown), inclusion bodies in the hepatocytes were observed in the proband's liver. Several hepatocytes had PAS-positive with diastase-resistant inclusion bodies (Fig. 3A) and immunoreactive substances with anti-alAT antibody (Fig. 3B) especially in the periportal regions, suggesting the existence of aggregated alAT molecules in hepatocytes. No such inclusion bodies were recognized in the hepatocytes from normal liver biopsy specimens under the same staining conditions.

Identification of Single-base Substitution in alAT-coding
Exons-Direct sequencing of all coding exons (IC, 11, 111, IV, and V) of the alAT gene using single-stranded DNA from asymmetric PCR revealed a single-base substitution in exon I1 of the a1AT gene. In this context, the proband showed a homozygous C to T mutation resulting in replacement of Ser"j (TCC) by Pheh3 (TEC) (lane 3, Fig. 4). His father (lane 2, Fig.  4),mother, sister, and daughter (not shown) were all heterozygous with both C and T at this base position. These results correlated with the fact that the serum alAT levels detected in the family members were about half the normal serum level of healthy individuals while the level of alAT of the proband was severely low (Fig. 1). Together with the data for serum alAT concentration and the base substitution, it is likely that this missense mutation is responsible for the deficient state in a1AT tiiiyama. When compared with the reference sequence of M1 (Ala"I3) (16,17), residue 213 of Siiyams was Val, indicating that it is derived from the normal variant a1AT M1 ( V a P ) gene.
alAT mRNA Transcripts by Northern Analysis-As the alAT transcript is known to exist in peripheral blood leukocytes (18,19), the mRNA level of alAT S i i , , , , in leukocytes were determined by Northern analysis. Similar alAT mRNA transcript levels were observed in the proband and normal subjects (Fig. 5), indicating that transcription and the stability of mRNA transcripts of the deficient a1AT Siigama variant are unaffected.

DISCUSSION
Serpin is the name given to a superfamily of proteins that are inhibitors of specific serine proteases (1, 6, 7). Serpins in mononuclear phagocytes and gray region corresponds t,o signal peptides. The region indicated by an arrow is shown below in the sequencing gel. Sequencing was performed by the didexoy termination method using second PCR products as t,emplate (for details, see "Materials and Methods"). Analysis of all coding exons of the crlAT gene revealed the single abnormality in exon 11. Shown in the sequence from Ile'" to Ser ' a1 -Antitrypsin Siiyomo include plasma protease inhibitors such as alAT, al-antichymotrypsin, antithrombin 111, plasminogen activator inhibitor, and even ovalbumin and protein Z (1,6,7,20). The existence of this superfamily derived from analyses of amino acid homology by Hunt and Dayhoff (21). They indicated that highly similar regions exist among alAT, antithrombin 111, and ovalbumin. The term serpin was proposed by Carrell and Travis (7) from comparisons of the sequences in the reactive centers of these inhibitors. The serpins have developed by divergent evolution over a period of some 500 million years. During their profound evolution, they have acquired specialized inhibitory diversity against their cognate proteases, and others, for example, thyroxin-and cortisol-binding globulins or ovalbumin, are presumed to have lost their function as serine proteinase inhibitors. The most researched and studied serpin is a1AT. The prime physiological role of a1AT is the inhibition of elastase derived from leukocytes. The clinical consequence of alAT deficiency in serum is the early onset of emphysema, a destruction of lung parenchyma by neutrophil proteases accumulated in the lung by smoking (2-4, 11). Structural understanding of the serpins was accelerated by the crystallographic structure of alAT (22). Huber and Carrell (1) recently analyzed the threedimensional structure of alAT and discussed the structure and function of the serpins. They listed 51 conserved amino acid residues in the serpin superfamily. Among them, four have already been reported in natural serpin variants with pathological significance, including Glu"' + L Y S~~' in a Ztype alAT (16, 23) and G~u~~~ + VaP4 of an S-type alAT (8). The most interesting mutant which occurs naturally is Pro"69 + Leus6' in alAT Mheerlen which occurs at the beginning of sheet 4B (24). When the mutation was evaluated in the background of the serpin structure, the mutation at the identical position in the crystallographic structure was already reported in antithrombin I11 Utah (Pro407 + Leu407) causing reduction in the serum level and increase in the tendency to thrombosis (25). This example indicates the validity of the alAT crystallographic structure.
The mutation found in alAT Siiysma, Sers3 + Phe", occurs on one of the 51 residues noted as conserved by Huber and TABLE I Implication of amino acid substitutions in deficient, null, dysfunctional, and normal aIAT variants: relation to mutational matrix number and consensus serpin backbone The amino acid substitutions were categorized in relation to the consequence of its own substitution, that is deficient, null, dysfunctional, and normal variant. They were analyzed further in regard to the mutational matrix number (mmn) and the mutated position of the conserved residue of the serpin superfamily backbone. Alignment of amino acid sequences of 20 members of the serpin superfamily based on the construction of this  (27). Briefly, 0 means neutral, +4 means 2.5 times as frequent, and -4 means 0.4 times less frequent as average. "The definition of "consensus" is after Ye et al. (20) where a residue is observed if present in half or more sequences, and a plus "+" is assigned if conserved residues are present in two-thirds or more sequences. "Conserved" means positive numbers in the mutational matrix table (27) for a given amino acid substitution using human nlAT (M1 (Val?")) as standard.

TABLE I1
Amino acid difference between human (MI (Val","II and baboon uIAT: relation to mutational matrix number and consensus serpin backbone Amino acid differences between human (Ma (Val":')) and baboon lvlAT (26) were sorted in regard to the mutational matrix number. Carrell (1). In this respect, a l A T Siiyama of this study is unique in the following points. First, mutation at Ser"j has a profound effect on the three-dimensional structure of the alAT molecule because 0' of Ser"" initiates helix B, hydrogen bonded to N of Ser"6, and stabilizes a portion of sheet 5B by bonding to 0 of Leu''f13. It also participates in the folding of the internal core which consists mainly of the most conserved secondary structural elements (helix B, sheet 3A, sheet 4B, sheet 5B). It can be assumed that the changes in properties resulting from the replacement of hydrophilic Sers3 by hydrophobic Phe" may influence the integrity and organization of the alAT molecule. This is related to the marked cathodal shift to S on IEF (Fig. 2) and the existence of immunoreactive aggregates in haptocytes from the proband (Fig. 3). The intracellular events caused by the altered protein structure eventually results in the reduction in the serum level of alAT. As summarized by Huber and Carrell (l), there is a convincing correlation between the structural changes predicted and the actual functional consequences observed. a l A T is one of the most extensively studied human proteins in regard to amino acid replacement, because of its pleomorphic nature and the clinical consequences caused by deficiency in serum. In addition, amino acid differences between human alAT and baboon a1AT (26) are evolutionally interesting. When these amino acid substitutions are analyzed in relation to the mutational matrix number (mmn) proposed by Dayhoff et al. (27) there is a distinct correlation between the pathological substitutions and the substitutions found in the physiological variants (Table I) or in baboon a l A T (Table 11). Replacements with more negative (zero to minus) mutational matrix numbers at the positions of highly conserved residues are found in the variants of pathological importance, suggesting evolutionally rare and functionally diverse substitutions occur in the sterically critical area. In the case of Siiy,,,, the mutation occurs at conserved residue Ser"j (negative mutation matrix number -3 (Ser-Phe)). In contrast, nine normal variants and 29 amino acid differences between human a l A T and baboon a l A T occur at less conserved residues with more positive (zero to plus) mutational matrix number. Although one difference (Ile"'9 + VallGY) between human and baboon occurs a t a conserved position, the mutational matrix number of Ile to Val is +4, which is one of the most frequent substitutions found in evolutional change (27).