Transglutaminase 1 Mutations in Lamellar Ichthyosis
LOSS OF ACTIVITY DUE TO FAILURE OF ACTIVATION BY PROTEOLYTIC PROCESSING*

Eleonora CandiDagger , Gerry Melino§, Armin Lahm, Roberta CeciDagger §, Antonello RossiDagger §, In Gyu Kimparallel , Barbara Ciani§, and Peter M. SteinertDagger **

From the Dagger  Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, § Istituto Dermopatico Dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, and Biochemistry Laboratory, University of Rome Tor Vergata, and University of L'Aquila, Rome, 00133 Italy, the  Department of Biological and Chemical Computation, Istituto di Ricerche di Biologica Molecolare P. Angeletti, Pomezia, 00040 Italy, and the parallel  Department of Biochemistry, Seoul National University, Seoul 449-900, Korea

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Lamellar ichthyosis is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. It is caused by mutations in the TGM1 gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGase 1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGase 1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To understand better how point mutations, deletions, and truncations found in lamellar ichthyosis disease affect the structure and function of TGase 1, we have expressed in baculovirus and keratinocytes a number of reported TGase 1 mutants. The structural implications of these mutations were examined using a homology-derived three-dimensional model of TGase 1 generated from the known x-ray structure of the related coagulation factor XIIIa enzyme. The present studies demonstrate that loss of TGase 1 activity is not restricted to mutations that directly affect the enzymatic activity. We report a new class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Congenital recessive ichthyoses (CRI)1 describe a clinically heterogeneous class of disorders of cornification that primarily affect the epidermis and hair (1-6). The phenotype appears at birth often presenting as a translucent collodion membrane encasing the newborn but later progresses to a variety of clinical findings ranging between fine white scaling, often combined with erythroderma, to large plate-like brown scales without erythema. Other clinical findings include palmar-plantar hyperkeratosis, eclabium, ectropion, scarring alopecia, and diminished skin barrier function. Recent studies have revealed marked biochemical (7, 8) as well as genetic variations in CRI patients as follows: to date, in certain families, linkage has been described to chromosome regions 14q11 (9, 10), 2q33-35 (11), or to neither (11-13). However, all cases of the large brown plate scaling phenotype have been linked to 14q11, and several different mutations in the TGM1 gene located at this site have been identified (10, 11, 14-15). The term lamellar ichthyosis (LI), the former umbrella name of the CRI group of diseases, should now be used exclusively for the CRI disease involving mutations in this gene (13).

The TGM1 gene encodes transglutaminase 1 (TGase 1) which is a member of a class of enzymes that form Nepsilon -(gamma -glutamyl)lysine or mono- or bis(gamma -glutamyl)spermidine isopeptide bond cross-links between proteins, thereby forming stable, insoluble macromolecular assemblies (16-19). TGase 1 is one of at least three such enzymes known to be involved in the formation of barrier function in stratified squamous epithelia, in particular in the formation of the cornified cell envelope, which is a 15-nm thick layer of protein deposited just inside the cell periphery (18, 20-23). The cornified cell envelope is formed when TGases 1 and 3 (and perhaps TGase 2) cross-link a variety of structural proteins including desmosomal proteins, involucrin, the small proline-rich proteins, and loricrin in the epidermis, trichohyalin in the inner root sheath of the hair follicle, as well as other undefined proteins in the hair fiber cuticle. In vitro studies have indicated that some substrates including loricrin and involucrin require both enzymes for apparent sequential cross-linking to form normal stabilized structures (22, 24). This requirement may explain the phenotype of LI disease; the TGase 3 enzyme cannot replace the essential cross-linking performed by the absent TGase 1 enzyme. The TGase 1 enzyme itself shows a complex pattern of activities since it exists in multiple cytosolic and membrane-bound forms in terminally differentiating keratinocytes (25, 26). However, whereas much of the enzyme remains in the intact, very low specific activity (zymogen) form of about 106 kDa, most activity resides in a proteolytically processed form of 67/33/10-kDa chains that are held together by secondary interactions while bound to the membrane through acyl myristate and palmitate adducts on the 10-kDa portion (26, 27).

Mutations in the TGM1 gene that produce a defective enzyme due to truncations, point substitutions, etc. are located on a number of sites along the protein. Patients can be either homozygous for a single mutation or compound heterozygous for two different mutations (10, 11, 14-16). However, it is not yet clear how these changes affect the structure and function of TGase 1. To explore this, we have expressed in baculovirus and keratinocytes a number of reported mutant TGase 1 forms to study their activity and processing properties. Also we have examined the likely structural consequences of the mutations by comparison with the known three-dimensional structure of the related factor XIIIa (fXIIIa) enzyme.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of the Recombinant Transfer Vectors and Recombinant Baculoviruses in Insect Cells-- All recombinant DNA technology was done according to standard procedures (28, 29). A full-length wild type human TGase 1 cDNA (30) was cloned into the baculovirus vector pVL1392 (PharMingen) by insertion at the EcoRI sites. This was then used as a template for the eight mutant forms (see Table I) that correspond to mutations seen in LI patients selected in this study. The TransformerTM Site-directed Mutagenesis Kit (CLONTECH) based on the method described by Deng and Nickoloff (31) was used for introduction of mutations.

The TGase 1 cDNAs in the pVL1392 vector were under the transcriptional control of the strong baculovirus polyhedrin promoter. The nine TGase 1 recombinant virus clones (wild type and eight mutant forms) were obtained by co-transfection of each vector with the modified Autographa californica nuclear polyhedrosis virus DNA (BaculoGold DNA, PharMingen). BaculoGold DNA carries a lethal deletion and does not code for viable virus particles by itself. Co-transfection of this DNA with a complementing plasmid construct rescues the lethal deletion of this virus DNA and reconstitutes, by homologous recombination, viable virus particles inside the transfected insect cells (32). Insect cells (Sf9, PharMingen) were grown in Grace's insect medium supplemented with 10% fetal calf serum (Life Technologies, Inc.).

In the co-transfection experiments, 2 × 106 cells were plated in 60-mm dishes. Two µg of each of the nine vectors was separately mixed with 0.5 µg of linearized viral BaculoGold DNA and incubated for 10 min at 23 °C. After incubation, 1 ml of transfection buffer (25 mM HEPES, pH 7.1, 125 mM CaCl2, 140 mM NaCl) was added to each tube and mixed. The DNA/transfection buffer mixtures were added to plates that contained 1 ml of regular medium and incubated at 27 °C for 3-4 h. At the end of the transfection, the medium was replaced with fresh Grace's insect medium, and the cells were kept for 5 days at 27 °C. The medium containing the nine recombinant virus clones was amplified to produce a high titer stock solution, and the titers (1-8 × 108 plaque-forming units/ml) were evaluated by end point dilution. For protein production, cells were maintained and infected, either as monolayer or in suspension, in a rotary shaker, with 1-4 × 108 plaque-forming units/ml of culture at a density of 1.5-2 × 106 cells (>95% viability) per ml of culture. Expression of the nine recombinant TGase 1 clones was confirmed by Northern blot analysis. Protein production was detected by Western blot using a specific polyclonal TGase 1 antibody (33). For lipid modification experiments, Sf9 cells were metabolically labeled (1 mCi each/ml of medium) with [14C]palmitate or [14C]myristate (NEN Life Science Products, 650 Ci/mol). The labels were added 4 h prior to harvesting.

Construction of Mammalian Expression Vectors-- The wild type and four mutant TGase 1 cDNA clones (R141H, R322Q, S41Y, and R314L) were excised at the NotI and XbaI sites from pVL1392 vector and directly subcloned into the mammalian expression vector pCMV (CLONTECH), under the control of the human cytomegalovirus immediate enhancer/promoter. In addition, a tag of histidine residues had been attached to the carboxyl-terminal end of the wild type and the two active mutant TGase 1 clones (S41Y, R314L). We generated these clones by polymerase chain reaction amplifying with standard conditions (34) from TGase 1 cDNA vectors used for baculovirus expression. The polymerase chain reaction primers were designed with an extension of 16 nucleotides containing NotI site at the 5'-end (plus primer, 5'-ATAAGAATGCGGCCGCATGGATGGGCCACGTTCCGATGTGGGCCGTTG-3') and an extension of 18 nucleotides that encode for six histidine residues followed by a termination codon TGA and BamHI site at the 3'-end (minus primer, 5'-TGCTCTAGACTAATGATGATGATGATGATGAGCTCCACCTCGAGATGCCATAGGGA-3'). The modified clones were sequenced to verify amplification efficacy and then assembled into the mammalian expression vector pCMV for transfection studies. These cultures were labeled with [35S]cysteine/methionine as described below since we found that the polyhistidine monoclonal antibody (Sigma) also immunoprecipitated processed forms of profilaggrin and filaggrin that are expressed in cultured NHEK cells in differentiating conditions; these proteins do not contain cysteine or methionine (35).

Cell Culture, Transfections, beta -Galactosidase Activity, and Protein Assay in Keratinocytes-- Cryopreserved normal human epidermal keratinocytes (NHEK) were obtained from CLONTECH (San Diego, CA) and grown in calf skin collagen (Sigma)-coated dishes in serum-free keratinocyte growth medium (KGM, Clonetics) at 0.05 mM Ca2+, supplemented with 60 µg/ml bovine pituitary extract. Third passage cells were used for transfection experiments. Transient transfections were performed in triplicate using Lipofectin reagent (Life Technologies, Inc.), as described previously (36). Briefly 2-3 × 105 cells were plated in each 35-mm well of six-well culture plates 16-20 h before transfection. Transfections were done when cultures had reached 70% confluency. Transfection efficiencies were always monitored by use of a cytomegalovirus beta -galactosidase construct (pCMV-beta , CLONTECH). Cells were washed once at 37 °C with phosphate-buffered saline and then preincubated for 30 min at 37 °C with keratinocyte serum-free medium. For each well, 1.5 µg of reporter plasmid and 0.5 µg of pCMV-beta were mixed with 6 µg of Lipofectin and incubated for 20 min at 23 °C. The lipid/DNA mixture was then added into each well and incubated for 3-4 h. After that, medium was removed, and to improve transfection efficiencies, cells were shocked with 15% glycerol in keratinocyte serum-free medium for 3 min. Medium containing either 0.05 or 1.2 mM Ca2+ was then replaced. Some transfection experiments were performed in 60- or 100-mm dishes, with proportional increase in seeding densities, amounts of plasmids, and Lipofectin. In some cases cells were harvested 50-120 h post-transfection, and in labeling experiments 6 h prior to harvesting, 2 µCi/ml of a mixture of [35S]cysteine and [35S]methionine (Amersham Pharmacia Biotech, 14.3 mCi/ml) was added. In one set of experiments to measure half-lives, this amount of isotope was added to the cultures immediately following transfection (25-27) for 6 h, and then the medium was replaced with high Ca2+ medium, and at different time points cells were harvested.

Cells were lysed in 0.2 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and a mixture of protease inhibitors (Boehringer Mannheim) that included 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml benzanidine, 5 µg/ml pepstatin, 5 µg/ml aprotinin), and the suspension was homogenized by sonication. The samples were centrifuged for 15 min at 14,000 × g at 4 °C to separate the cytosolic and particulate fractions. The particulate fractions were washed with lysis buffer, and then membrane-bound proteins were extracted into lysis buffer containing 0.1% Triton X-100 in ice for 30 min. After centrifugation, solubilized proteins were separated by membrane debris and washed with lysis buffer to remove the detergent (37).

Aliquots of the cytosolic and membrane-bound fractions were used for TGase assay, TGase immunoprecipitation, column chromatography, and for protein quantification, using methods described previously (25-27). The beta -galactosidase activity was assayed by using a commercial enzyme assay system (Promega). Where necessary, TGase activities were normalized by protein content and beta -galactosidase activity. The final data are the average of at least three independent experiments, each with duplicate samples.

Cross-linked cell bodies were harvested following exhaustive extraction in SDS and 2-mercaptoethanol as for the preparation of cornified cell envelopes (20, 21), resuspended in 1 ml of phosphate-buffered saline, and light scattering at 310 nm was measured.

In Vitro Cross-linking Experiment-- Membrane-bound TGase 1 was isolated from the particulate fraction of Sf9 cells exactly as described above. Recombinant 35S-loricrin was used as a complete substrate as described previously (24). In the cross-linking experiment 25 µg of labeled loricrin (about 8 × 104 dpm) was utilized in a 100-µl reaction volume, and the amount of recombinant baculovirus TGase 1 that incorporated 0.7 pmol/min [14C]putrescine (Amersham Pharmacia Biotech,, 118 mCi/mmol) into casein was used. The reaction was initiated by the addition of 10 mM CaCl2. Aliquots were stopped by addition of 10 mM EDTA after 15, 30, 60, and 120 min. The cross-linked products were separated on a 4-12% polyacrylamide gels and analyzed by autoradiography.

Generation of the TGase 1 Model-- The three-dimensional model of human TGase 1 was generated on the basis of the known atomic structure of human factor XIIIa (fXIIIa) as present in the Protein Data Base (38) entries 1GGT and 1FIE (39, 40). By using the modeling packages WHATIF (41) and INSIGHT-II (38, 42), the sequence of TGase 1 residues 107-789 was inscribed onto the structural frame provided by fXIIIa residues to 43-729. By using loop-search and debumping options, the resulting model for TGase 1 was then optimized at positions where deletions (Ser125, Gly187, and Pro505) or non-conservative substitutions occurred, respectively. Residues Gly96-Gly106 of TGase 1 including the 97NAAGDG102 hexapeptide were modeled manually onto residues Asn18-Glu30 of the fXIIIa activation peptide. Pictures were generated with the INSIGHTII software.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutant TGase 1 Forms Studied-- The purpose of the present work was to explore the structural-functional consequences of some of the known mutations of the TGM1 gene seen in LI. Data from protein expression and transfection studies were combined with structural information from a predicted three-dimensional model of the TGase 1 enzyme based on the known crystal structure of the related factor XIIIa enzyme. The TGase 1 mutations studied in the present work are listed in Table I, and as our data show, they offer a spectrum of alterations affecting the enzyme and afford new insights into the molecular basis of LI disease.

                              
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Table I
Mutant forms of TGase 1 studied in this paper

Expression and Biochemical Characterization of Recombinant Human Wild Type and Mutant Forms of TGase 1-- We expressed in the baculovirus system the human wild type TGase 1 cDNA and the eight mutant forms that have been reported in LI which are listed in Table I. Protein expression was evident from 48 h postinfection and reached a maximum at 72 h (Fig. 1, A and B). Western blot analyses using a specific polyclonal anti-human TGase 1 antibody revealed that the recombinant enzymes were not proteolytically processed in this system and remained as the full-length protein with an apparent molecular mass of 97 kDa (Fig. 1B). The mutation M422stop generated a truncated protein of size about 55 kDa (Fig. 1, A and B). As has been observed in NHEK cells, the baculovirus-expressed TGase 1 (wild type and mutant forms) was present in both the cytosol and membrane-bound fractions. About 90% of each of eight recombinant proteins was retained in the latter (data not shown), which is very similar to the distribution of the TGase 1 enzymes in proliferating basal epidermal keratinocytes or stationary NHEK cells grown in low Ca2+ submerged liquid cultures (26). The one exception was mutant form S41Y, in which about half of the protein was present in the cytosol (see Fig. 4F). The recombinant baculovirus-expressed wild type TGase 1 was also modified by lipids (Fig. 1C). The labeling of the cytosolic and membrane-bound enzyme forms with myristate indicates the protein was constitutively N-myristoylated as in keratinocytes (27). The weak incorporation of palmitate also implies partial S-acylation by myristate and palmitate. Similar data were obtained for the R141C and R314L mutants (data not shown), suggesting their distributions onto the membrane fraction were due to normal wild type lipid modification. However, in the case of the S41Y mutant, there were only traces of lipid incorporation, consistent with increased amounts in the cytosolic fraction. Furthermore, the recombinant wild type enzyme was capable of cross-linking in vitro its natural substrate loricrin (Fig. 1D) with a pattern of polymerization similar to that described previously for the bacterially expressed enzyme (24). Together, these data indicate that the wild type TGase 1 enzyme expressed in baculovirus has properties very similar to the native enzyme expressed in NHEK cells. Moreover, we found that the wild type recombinant baculovirus enzyme has an activity half-life of about 12 h (data not shown). Thus it is much more stable than the enzymes isolated from bacteria (34) or keratinocytes (24-26, 44) and is well suited for in vitro cross-linking studies.


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Fig. 1.   Expression and characterization of wild type and mutant forms of TGase 1. A, Coomassie staining of the 30 µg of total protein from Sf9 cells infected with wild type baculovirus (CTRL) and recombinant baculoviruses bearing wild type TGase 1 and the eight mutants cDNAs as shown. B, Western blot using a specific polyclonal antibody for TGase 1; the asterisks denote the TGase 1 protein forms of about 97 or 55 kDa. C, autoradiography of Sf9 cells infected with wild type baculovirus (ctrl) or recombinant baculovirus bearing wild type TGase 1 labeled with [14C]palmitic and [14C]myristic acid in the cytosolic and membrane-bound fractions. D, in vitro cross-linking of 35S-labeled human loricrin by membrane-bound recombinant baculovirus TGase 1, in the presence of EDTA (ctrl) of after reaction for the times shown. As for the bacterially expressed enzyme (24), both oligomerization and intrachain cross-linking occurs.

Specific Activities of Recombinant Wild Type and Mutant Forms of Human TGase 1-- Table II lists the specific activities of the cytosolic and membrane-bound forms of the wild type and eight mutant recombinant enzymes. The very low specific activities of the wild type enzyme were almost identical to those of the native intact (106-kDa form) enzyme expressed in NHEK cells or isolated foreskin keratinocytes (26). As expected, most analyzed mutants showed reduced enzymatic activities. Both membrane-bound and cytosolic TGase 1 activities were reduced to 0-20% of the wild type control for mutations R141H, R142H, R141C, and L268Delta . The specific activity of R322Q was also reduced. The truncated enzyme generated by the premature stop codon (M442stop) was inactive, as expected from earlier deletion cloning experiments (34). Unexpectedly, two mutant forms (S41Y and R314L) showed 2-4-fold increases in specific activities in comparison to the wild type enzyme (Table II).

                              
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Table II
Specific activities of wild type and mutant TGase 1 forms expressed in baculovirus
Specific activities are expressed as picomoles of [14C]putrescine incorporated into succinylated casein/h/pmol of TGase 1 protein. TGase 1 amounts are estimated by active site titriation with [14C]iodoacetamide (34). The results are averages ± S.D. of 3-5 measurements.

Transfections of Wild Type and Mutant Forms into NHEK Cells-- The full-length wild type and four mutant cDNAs (two of low or zero activity, R141H and R322Q, and two of higher activity, S41Y and R314L) were transfected into NHEK cells. Transfection efficiencies within the range of 15-30% were obtained in these experiments as estimated by co-transfection with a beta -galactosidase control vector (data not shown; and see Fig. 3C). Each activity value was standardized for units of beta -galactosidase in order to compare the different transfection experiments with the different constructs. These experiments were performed under proliferating (in low Ca2+, 0.05 mM) and differentiating (in high Ca2+, 1.2 mM) conditions (Fig. 2, A and B, respectively). As expected for NHEK cells (26, 27), in both cases most of the transfected TGase 1 enzyme was directed to the membrane fraction. In low Ca2+ media (Fig. 2A), while the activity level of the wild type transfectant cultures increased commensurate with the transfection efficiency, the activities of the R141H and R322Q mutants were similar to those of the untransfected or sham-transfected NHEK cells, indicating they are not functional, as observed in baculovirus (Table II). Likewise, the two active mutants (S41Y and R314L) generated total levels of enzyme activity that were higher than for the wild type construct and commensurate with the transfection efficiencies, indicating they were also as functional in NHEK cells as in baculovirus. However, in high Ca2+ media, the level of activity of each of the mutant constructs was at background level and lower than for the wild type construct (p < 0.01) (Fig. 2B). Similar results were obtained from the measurement of the formation of cross-linked cell bodies in the same cells in that the data paralleled the activity levels (data not shown).


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Fig. 2.   Transfections of wild type and mutant TGase 1 forms into NHEK cells grown in low and high calcium. NHEK cells were transfected in low calcium (0.05 mM) and grown in low calcium (A) or high calcium (1.2 mM) (B) media for 48 h, using the wild type TGase 1 and the four indicated mutant forms. Total TGase activity was measured for membrane-bound (closed bars) and cytosolic (stippled bars) fractions and standardized for units of beta -galactosidase activity. Lane C indicates the endogenous TGase 1 activity (keratinocytes were sham-transfected only with the beta -galactosidase control vector). All data are the averages (±S.D. of 3-5 experiments).

The S41Y and R314L Mutant TGase 1 Forms Are Not Proteolytically Processed in Terminally Differentiating NHEK Cells-- To explore further these differences, new constructs of the wild type and S41Y and R314L mutant TGase 1 forms were assembled in a mammalian expression vector system in which a tag of six histidine residues had been attached to the carboxyl-terminal end. This was done in order to distinguish between the endogenous and mutant TGases following transfection into NHEK cells.

In initial experiments, we tested whether the presence of this tag interferes with the expression and/or processing of the wild type TGase 1 protein in differentiating NHEK cells grown in the presence of [35S]methionine/cysteine. The total (combined cytosolic and membrane-bound) TGase 1 forms present in the cells including the transfected and endogenous enzymes were immunoprecipitated with the polyclonal anti-TGase 1 antibody (Fig. 3, A and B) and resolved by fast protein liquid chromatography into two major peaks that contained the full-length TGase 1 protein of about 106 kDa (fraction 84) and the processed complex of 67/33/10-kDa bands of TGase 1 (fraction 94), respectively. Essentially all activity was attributable to the latter, as described previously (26, 27) (Fig. 3B). Other minor peaks of activity in fractions 36, 66, and 74 contain the cytosolic 67-, 106-, and 67/33-kDa complex components, whereas fraction 120 contains the inactive 33-kDa band (which is poorly precipitated by the TGase 1 antibody (25, 33)). Immunoprecipitation of the same cultures with the polyhistidine antibody generated identical data (Fig. 3, C and D), except that the amount of label precipitated was about 28% that with the TGase 1 antibody, which thereby provides an estimate of the transfection efficiency. Moreover, the specific activities of the highly active processed forms of fractions 94 were the same (1050 ± 180 and 980 ± 150 pmol of [3H]putrescine/h/pmol TGase 1 protein, respectively) and are almost 200-fold greater than that of the intact 106-kDa form (Table II; Ref. 26). Samples of the immunoprecipitates of both antibodies were resolved on gradient SDS-PAGE gels (Fig. 4, B and E). The results obtained were the same and demonstrated that in both cases about half of the total TGase 1 protein was processed into the 67-, 33-, and 10-kDa forms. Thus the presence of the histidine tag on the carboxyl terminus does not interfere with TGase 1 processing or activity.


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Fig. 3.   The S41Y and R314L mutant forms of TGase 1 are not proteolytically processed in terminally differentiating keratinocytes: chromatography study. Mammalian expression vector constructs containing the His-tagged wild type and the two mutant TGase 1 forms shown were transfected into NHEK cells and grown in high calcium media for 4 days. Following a 6-h pulse with [35S]methionine/cysteine prior to harvesting, the total (cytosolic and membrane-bound) TGase 1 forms were immunoprecipitated with either the TGase 1 (A and B) or polyhistidine (C-H) antibodies, chromatographed by fast protein liquid chromatography, and assayed by measurement of either 35S dpm (left columns: dpm/fraction) or TGase activity (right columns: dpm of [14C]putrescine incorporated into succinylated casein/fraction).


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Fig. 4.   SDS-PAGE gels reveal absence of proteolytic processing of S41Y and R314L mutant forms of TGase 1. Cultures similar to above were harvested at the times indicated, processed as in Fig. 3, but analyzed by SDS-PAGE on 10-20% gradient gels and autoradiographed for 4-16 days. Immunoprecipitated with the following: A-C, TGase 1 antibody; D-G, polyhistidine antibody. A and D, non-transfected NHEK cultures; B and E, transfected with the wild type TGase 1 construct; C and F, transfected with the S41Y construct; and G, transfected with the R314L construct. The sizes of the processed TGase 1 protein components are shown. Note that in these experiments, much of the 10-kDa band (arrowhead) was lost by apparent degradation.

These experiments were repeated with NHEK cells transfected with the constructs containing the His-tagged S41Y and R314L mutant TGase 1 forms. By using identical culture conditions as above, chromatography (Fig. 3, E-H, respectively) and SDS-PAGE gels (Fig. 4, F and G, respectively) revealed that the products immunoprecipitated by the polyhistidine antibody correspond to the intact membrane-bound 106-kDa form. Therefore, for both mutants, only trace amounts had been proteolytically processed under these terminally differentiating conditions.

In pulse-chase experiments we estimated the half-lives of the intact wild type and S41Y and R314L mutant forms. Whereas the half-life of the membrane-bound wild type protein was about 30 h as expected (27), the half-lives of the S41Y and R314L mutants were clearly greater (50 and 60 h, respectively) (Fig. 5A). Similarly, the cytosolic forms are more stable (40 and 60 h, respectively, versus 20 h for the wild type) (Fig. 5B).


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Fig. 5.   The S41Y and R314L mutant TGase 1 forms are more stable than the wild type enzyme. Cultures similar to Fig. 3 were pulsed for 6 h with high calcium media containing [35S]methionine/cysteine following transfection, washed, and then grown for up to 4 days in high calcium media. Membrane-bound (A) and cytosolic (B) proteins were immunoprecipitated with the polyhistidine antibody, resolved by SDS-PAGE as in Fig. 4, and exposed for quantitation by scanning densitometry.

Together, these data demonstrate that although these two mutants are stably expressed and retained in the NHEK cells, they remain as the intact very low specific activity (zymogen) proteins. They are not processed into the highly active functional enzymes as for the wild type protein.

Predicted Structural Model of TGase 1-- In order to explore the structural consequences of the mutant forms studied in this paper, we constructed a three-dimensional model of human TGase 1 (Fig. 6) using as the basis the known crystal structure of the related human fXIIIa (39). The high sequence conservation and few deletions provide a realistic model of the TGase 1 molecule. This includes four main domains that have been designated the amino-terminal beta -sandwich, the central core domain containing the active site (catalytic core domain), and beta -barrels 1 and 2 (39). In the case of fXIIIa, an activation peptide precedes the beta -sandwich domain. In the case of TGase 1, a approx 90-residue sequence that is involved in membrane anchorage and substrate recognition or specificity (26) precedes the beta -sandwich domain. We note that when TGase 1 is proteolytically activated to generate the 10/67/33-kDa complex form, the sites of cleavage are located near the junction of the membrane anchorage and beta -sandwich domains and to the junction of the catalytic core domain and beta -barrel 1 domain, respectively (Fig. 7). Since both the full-length 106-kDa TGase 1 and the 67/33/10-kDa complex normally purify in monomeric form (Refs. 25-27; see Fig. 3), only one monomer of the dimeric fXIIIa structure was used in the modeling study, omitting the part of the fXIIIa activation peptide preceding Asn18, the part mediating the intermolecular inhibition (Figs. 6 and 7) (39).


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Fig. 6.   Three-dimensional model of TGase 1. View of the entire model with the four main structural domains and proposed interaction between the 10-kDa membrane anchorage fragment with the body of TGase 1. Most mutations described here are located at the interface between the amino-terminal beta -sandwich domain and the catalytic core domain. Spheres indicate the positions of the mutated residues. TGase 1 residues 95-106 are highlighted in orange. The 97NAAGDG102 hexapeptide, tightly associated with the beta -sandwich and catalytic domain, is magenta. The association of the 10-kDa membrane anchorage fragment with the main body of the enzyme is proposed to occur in the region between the beta -sandwich domain and the active site around Cys375, in a conformation that inhibits TGase 1 activity. On proteolytic cleavage at Arg92 and formation of the active 10/67/33-kDa complex, the 10-kDa fragment undergoes structural re-arrangements but remains attached through specific interactions.


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Fig. 7.   Alignment of the amino-terminal portions of fXIIIa and TGase 1. The two TGase 1 hexapeptide motifs (55NAADDD60 and 97NAAGDG102) are highlighted by gray boxes. The major proteolytic cleavage site in TGase 1 (Arg92, large arrowhead) precedes the amino-terminal boundary of the beta -sandwich domain by 14 residues. The corresponding proteolytic activation site for fXIIIa is shown by the small arrowhead and is located in a portion of sequence (lowercase letters) that was not observed in the crystal structure (39). The fXIIIa sequences preceding Asn18 are shown in italicized lowercase letters. Black and white boxes delineate alpha -helix and beta -strand secondary structure features, respectively. The location of the S41Y substitution, acyl lipid attachment sites, and other sites of sequence identity are indicated.

Following proteolytic activation at Arg92, most of the membrane-bound TGase 1 remains associated with the 10-kDa membrane anchorage domain (26, 27) to form a 67/33/10-kDa complex. In the crystal structure of Ca2+-activated and thrombin-cleaved fXIIIa, the activation peptide remains associated with the body of the enzyme and adopts a practically identical conformation as compared with the fXIIIa zymogen (39, 40). In particular, we note a high homology between two hexapeptide segments of this domain of TGase 1, 55NAADDD60 and 97NAAGDG102 (Fig. 7), with a region of the fXIIIa activation peptide, 20NAAEDD25, that tightly interacts with the beta -sandwich domain and the catalytic core domain (39). This interaction is mediated by fXIIIa Asp243 and Arg252, both conserved in TGase 1 (Asp305 and Arg314). This suggests that the high overall structural similarity between these two enzymes also includes parts of the activation peptide of fXIIIa and the membrane anchorage domain of TGase 1. Based on the observed phenotype for the R314L mutant, we predict the second 97NAAGDG102 hexapeptide motif is associated with the amino-terminal beta -barrel domain in the unprocessed full-length 106-kDa form of the enzyme. The second motif, 55NAADDD60, might represent an alternative mode of binding, for example to modulate substrate specificity after cleavage at Arg92 (26, 34). Only the part of the fXIIIa activation peptide starting from Asn18 (corresponding to TGase 1 Gly95) was therefore included in the TGase 1 model. In the loop region following the 97NAAGDG102 segment, small local re-arrangements centered around 106GG107 were sufficient to close the loop leading to the first beta -strand of the beta -sandwich domain.

Mutant R322Q and Inactive Mutants R141C, R141H, and R142H-- As is evident from Fig. 6, most of the substitutions cluster around the interface between the amino-terminal beta -sandwich domain and the central catalytic core domain. An exception is R322Q near the surface of the catalytic domain far from the active site (Fig. 8A), consistent with its milder phenotype. Consequently, no particular role in either substrate recognition or enzymatic activity can be attributed to this residue. While this in principle is also true for positions Arg141 and Arg142, these residues, conserved in all members of the TGase family (30), are an integral part of a chain of hydrogen bonds connecting the amino-terminal beta -sandwich domain through Asp253 in the linker region to the catalytic core domain (Fig. 8B). Since both mutants showed expression levels and lifetimes comparable to that of the wild type enzyme in the baculovirus expression system, overall protein stability seems not to be affected. A conformational change affecting the relative orientation between the amino-terminal domain and the catalytic core domain is therefore the most likely cause for their enzymatic inactivity, as the amino-terminal domain is indispensable for substrate recognition and processing (26).


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Fig. 8.   Stereo images of the effects of the Arg141, Arg142, Arg314, and Arg322 mutations in the local environment. A, local environment around Arg314 and Arg322. Whereas Arg322 is exposed on the surface, Arg324 is buried in the domain interface and mediates (through Asp305) tight interactions between the catalytic domain and initial part of the 97NAAGDG102 hexapeptide motif (magenta). TGase 1 residues 95-96 preceding this motif are orange. Main chain functional groups involved in hydrogen bonds are shown in black. B, close view of the chain of hydrogen bonds connecting the amino-terminal beta -sandwich and the catalytic domains through Arg141, Arg142, and Asp253.

Deletion Mutants L268Delta and M442stop-- The absence of structural integrity, a prerequisite for most enzyme activity, readily explains the complete absence of catalytic activity in case of the M442stop mutation. Truncation of the polypeptide chain at this position removes a considerable part of the catalytic core domain (residues 443-572), presumably creating a misfolded central domain unable to assemble a functionally active site. Previous deletion cloning experiments documented loss of activity when these sequences were removed (34). As before for the R141H and R142H mutations, proper function of the enzyme will be destroyed due to the absence of a correctly oriented amino-terminal beta -sandwich domain.

Active Mutants S41Y and R314L-- Both of these mutants exhibited an increased basal activity of the unprocessed 106-kDa polypeptide and do not undergo activation by proteolytic cleavage at Arg92 in differentiating keratinocytes (Figs. 3 and 4). The relationship between the two phenotypes seems at first sight to be unrelated. Arg314 is located in the interface between the amino-terminal beta -sandwich domain and the catalytic core domain and mediates (through Asp305) specific interactions to the initial part of the hexapeptide motif of the 10-kDa fragment (Fig. 8B). Substitution of Arg314 into Leu would abolish these interactions and potentially destabilize the preceding loop region, as found for an analogous mutation in fXIIIa (R252I), which resulted in undetectable protein levels presumably due to reduced protein stability (45). However, the presence of Pro310 in the preceding loop region is likely to significantly increase the local conformational stability in TGase 1. At the corresponding position of fXIIIa, an aspartic acid (Asp248) is instead present. Presumably also, this increased stability, as reflected by a very long half-life (Fig. 5), prevents or interferes with proteolytic activation at the nearby Arg92.

For the S41Y mutation, no direct prediction can be derived from our model, since this residue is located in the membrane anchorage segment which could not be modeled. We suggest that this substitution promotes a conformational change in the 10-kDa fragment, which as for R314L, results in a net reduction of the inhibitory capacity of the 10-kDa fragment domain.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The TGase 1 enzyme exists in keratinocytes in multiple forms due to post-translational proteolytic processing (25-27). In proliferating or stationary cells, most protein exists as the intact form of low specific activity (zymogen) and is tightly bound to cellular membranes. (This form retains some activity and is not "inactive" as previously thought (26)). Only minor (<5-10%) amounts cycle into the cytosol. However, in terminally differentiating keratinocytes, up to 50% of the membrane-bound TGase 1 is proteolytically processed by cleavage at sites that correspond very closely to inter-domain junctions of its predicted structure. The resulting 67/33/10-kDa complex shows a 200-fold increase in specific activity and is responsible for most of the TGase 1 activity in keratinocytes during terminal differentiation (26).

In the present paper, we have explored the structure-function relationship for several mutants of the TGase 1 enzyme observed in LI disease. We have combined biochemical data obtained for expressed forms of the mutants with structural information from a homology-derived model of TGase 1. The mutant form M442stop results in large deletions of sequences known to be essential for activity. Apart from mutant R322Q, which has only a modestly reduced activity, most other mutants caused by point mutations (R141C, R141H, R142H, and L268Delta ) resulted in loss of function due to the failure to establish key ionic and hydrogen bond interactions (Fig. 8, A and B). These changes are likely to cause partial protein misfolding and/or domain rearrangements with resultant loss of activity, that is LI disease was caused by the greatly reduced enzyme activity in the keratinocytes.

Identification of a Novel Type of Mutant: Interference with Postsynthetic Proteolytic Activation-- For the last two mutants, S41Y and R314L, a more detailed characterization was necessary in order to understand why they should cause LI disease. At first glance they appear to involve gain of function; both mutant enzymes were significantly more active than wild type TGase 1 when expressed in baculovirus or transfected keratinocytes grown under proliferating conditions, in situations where only the unprocessed 106-kDa form of the enzyme is observed (Fig. 2 and Table II). Further experiments in terminally differentiating NHEK cells showed, however, that both variants could not be proteolytically processed into the functional highly active form (Figs. 3 and 4). In view of the large differences in specific activities between the unprocessed 106-kDa and the proteolytically activated 67/33/10-kDa complex (26), the net consequence of the mutant forms was therefore the same as for the loss-of-function mutants: insufficient enzyme activity for terminal differentiation.

We have shown in earlier deletion cloning experiments in bacteria that the 10-kDa membrane anchorage fragment dictates the catalytic state of the TGase 1 enzyme (26, 34). There are at least two possible ways how this could occur. One is the modulation of substrate specificity controlling the type of substrate that may approach the active site. Although no direct structural data are yet available, we propose a second possibility where at least parts of the membrane anchorage domain fold back onto the body of the TGase 1 enzyme so as to partially obstruct access to its active site. This model is reminiscent of what is observed to occur for the fXIIIa activation peptide, albeit intra-molecular instead of inter-molecular (Fig. 6). Our hypothesis is based on two findings. In the first, in vitro overlay experiments on the binding of different post-translationally modified 10-kDa domains showed that S-myristoyl and S-palmitoyl modifications of the cysteine-rich cluster (47CCGCCSC52) are required for interactions with the 67-kDa and 67/33-kDa components to form the highly active 67/33/10-kDa complex form of the TGase 1 enzyme (27). Second, in this study, we have recognized regions of high sequence homology of the membrane anchorage domain of TGase 1 (residues 97-102, and perhaps residues 55-60) with the region encompassing residues 20-25 of the fXIIIa activation sequence that is responsible for the inhibition of the fXIIIa enzyme. The contact points of this interaction involve specifically the catalytic domain residue Arg252 (fXIIIa) and Arg314 (TGase 1). Together, these data indicate the existence of rather specific interactions between the membrane anchorage and amino-terminal beta -sandwich/catalytic core domains of the TGase 1 enzyme. Thus in the R314L mutant, the substitution of Arg314 and failure to make key interactions (Figs. 6 and 9) partially releases the inhibitory effect of the membrane anchorage domain, resulting in a molecule of increased specific activity. Given the structural context around Arg314 in our TGase 1 model (Fig. 9), we furthermore conclude that this molecular event also increases the stability of the protein and interferes with proteolytic activation. Mutation of Arg314 into Leu is likely to affect Arg92 directly preceding the 97NAAGDG102 hexapeptide motif and, as a consequence, alter the accessibility to the cleavage site.


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Fig. 9.   Stereo images illustrating the effect of the R314L mutation on the interaction between Arg314 and the 97NAAGDG102 hexapeptide motif. Detailed view of the predicted interaction between the 97NAAGDG102 hexapeptide following the 10-kDa membrane anchorage domain and the body of the TGase 1 enzyme. Asp305 and Arg314 take the role of fXIIIa Asp243 and Arg252 in linking the fragment to the catalytic core domain. Mutation of R314 into Leu (L314, shown in green) will disrupt the chain of electrostatic and hydrogen bond interactions between the catalytic domain and functional backbone groups around Asn97. As a result, we predict that local structural changes occur affecting the conformation of residues preceding Asn97, including also Arg92, which is the site of proteolytic cleavage. For illustrative purposes a tentative position for Arg92 has been included in the figure.

In the case of the S41Y mutant form, a similar scenario has to be assumed, although in this case our model does not allow a direct correlation with structural information. However, we note the presence of an Arg-Arg dipeptide (35RR36) immediately upstream Ser41 that could in principle assume a function similar to fXIIIa Arg11/Arg12, which contact acidic residues near the active site thus blocking enzymatic activity (39). Conformational alterations caused by the presence of the substituted tyrosyl side chain could prevent the 10-kDa fragment to exhibit its full inhibitory effect and may also explain why the S41Y mutant form was less efficiently bound to membranes (Fig. 4).

Our data confirm the key role of the 10-kDa membrane anchorage domain for TGase 1 activity and highlight the sensitivity of the enzyme toward alterations affecting this region. Any mutation that disturbs the proper conformation of the 10-kDa membrane anchorage could therefore have drastic consequences for enzymatic activity or substrate specificity. More detailed structural information on this domain therefore seems warranted.

Altogether, the present studies demonstrate that loss of TGase 1 enzyme activity, which results in LI disease, can arise either from mutations that affect directly the correct folding of the protein or which instead affect the subsequent postsynthetic processing of the protein into its highly active functional forms. Accordingly, it is to be expected that LI disease can be caused by deficient TGase 1 activity due to adverse effects on other processing events, such as N-myristoylation, membrane anchorage due to failed lipid S-myristoylation, or S-palmitoylation, phosphorylation, etc. Furthermore, it is conceivable that mutations that affect directly those enzyme systems responsible for these post-synthetic modifications should result in a TGase 1 enzyme of greatly reduced specific activity or potential for proteolytic processing, with consequences of LI disease. In this regard, it is noteworthy that cases of CRI have been identified that do not involve mutations in the TGM1 gene (11-13). Further work must be done now to explore whether mutations in these ancillary gene systems, directly or indirectly affecting the TGase 1 enzyme, are the cause of CRI or LI disease.

During the preparation of this manuscript, another paper was published describing several new mutations of the TGM1 gene in LI, and structural modeling was performed to explain the diminished TGase 1 activity of the mutant forms (46). Many of the data and conclusions conform to our analyses described here. Two common mutant forms were analyzed. As in the present study, the mutant form S41Y was demonstrated to be less efficiently bound to membranes and to have a higher activity than the wild type (when assayed following transfection into keratinocytes derived from LI patients with no background TGase 1 activity). However, that study did not identify the observation discovered here that under differentiating conditions, the S41Y mutant form is likely to be disease-causing since it could not be processed into a highly active functional enzyme. Our study offers a possible explanation for this in the association of the membrane anchorage region of TGase 1 to the beta -sandwich domain. Similarly, that study described a mutant form R314C, which possessed low specific activity, presumably due to protein misfolding. In the present work, we show that the Leu substitution instead leads to an excessively stable protein that cannot be processed. Thus, biochemical analyses as performed in this study, coupled with activity and structural analyses, have provided more profound information about the properties of the TGase 1 enzyme system.

    FOOTNOTES

* The work was supported in part by Grant E413 from Telethon and the Neuroblastoma Association (to G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Laboratory of Skin Biology, NIAMS, Bldg. 6, Rm. 425, National Institutes of Health, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail: pemast{at}helix.nih.gov.

1 The abbreviations used are: CRI, congenital recessive ichthyosis; fXIIIa, factor XIIIa; LI, lamellar ichthyosis; NHEK, normal human epidermal keratinocytes; TGase 1, transglutaminase 1; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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