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• W2040258749 abstract "Type I transglutaminase (TG1) is an enzyme that is responsible for assembly of the keratinocyte cornified envelope. Although TG1 mutation is an underlying cause of autosomal recessive congenital ichthyosis, a debilitating skin disease, the pathogenic mechanism is not completely understood. In the present study we show that TG1 is an endoplasmic reticulum (ER) membrane-associated protein that is trafficked through the ER for ultimate delivery to the plasma membrane. Mutation severely attenuates this processing and a catalytically inactive point mutant, TG1-FLAG(C377A), accumulates in the endoplasmic reticulum and in aggresome-like structures where it is ubiquitinylated. This accumulation results from protein misfolding, as treatment with a chemical chaperone permits it to exit the endoplasmic reticulum and travel to the plasma membrane. ER accumulation is also observed for ichthyosis-associated TG1 mutants. Our findings suggest that misfolding of TG1 mutants leads to ubiquitinylation and accumulation in the ER and aggresomes, and that abnormal intracellular processing of TG1 mutants may be an underlying cause of ichthyosis. Type I transglutaminase (TG1) is an enzyme that is responsible for assembly of the keratinocyte cornified envelope. Although TG1 mutation is an underlying cause of autosomal recessive congenital ichthyosis, a debilitating skin disease, the pathogenic mechanism is not completely understood. In the present study we show that TG1 is an endoplasmic reticulum (ER) membrane-associated protein that is trafficked through the ER for ultimate delivery to the plasma membrane. Mutation severely attenuates this processing and a catalytically inactive point mutant, TG1-FLAG(C377A), accumulates in the endoplasmic reticulum and in aggresome-like structures where it is ubiquitinylated. This accumulation results from protein misfolding, as treatment with a chemical chaperone permits it to exit the endoplasmic reticulum and travel to the plasma membrane. ER accumulation is also observed for ichthyosis-associated TG1 mutants. Our findings suggest that misfolding of TG1 mutants leads to ubiquitinylation and accumulation in the ER and aggresomes, and that abnormal intracellular processing of TG1 mutants may be an underlying cause of ichthyosis. Transglutaminases comprise a family of multifunctional proteins that play an important role in protein stabilization and intracellular signaling (1Lorand L. Graham R.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 140-156Crossref PubMed Scopus (1223) Google Scholar, 2Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. J. Invest. Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The consensus view is that epidermal type I transglutaminase (TG1), 2The abbreviations used are: TG1transglutaminase type 1ERendoplasmic reticulumTMAOtrimethylamine-N-oxidem.o.i.multiplicity of infectionBiPbinding immunoglobulin proteinFCfluorescein cadaverineDMSOdimethyl sulfoxideEGFPenhanced green fluorescent proteinEVempty vector. which is expressed in surface epithelia, has an important and essential role in catalyzing protein-protein cross-link formation leading to formation of the cornified envelope (3Hennings H. Steinert P. Buxman M.M. Biochem. Biophys. Res. Commun. 1981; 102: 739-745Crossref PubMed Scopus (80) Google Scholar, 4Robinson N.A. LaCelle P.T. Eckert R.L. J. Invest. Dermatol. 1996; 107: 101-107Abstract Full Text PDF PubMed Scopus (60) Google Scholar, 5Steinert P.M. Candi E. Kartasova T. Marekov L. J. Struct. Biol. 1998; 122: 76-85Crossref PubMed Scopus (83) Google Scholar, 6Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 7Steven A.C. Steinert P.M. J. Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar, 8Yaffe M.B. Murthy S. Eckert R.L. J Invest. Dermatol. 1993; 100: 3-9Abstract Full Text PDF PubMed Google Scholar). The cornified envelope is a 15-nm thick structure comprised of covalently cross-linked proteins and lipids deposited adjacent to the inner surface of the plasma membrane in differentiating keratinocytes (7Steven A.C. Steinert P.M. J. Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar, 9Matoltsy A.G. J. Invest. Dermatol. 1976; 67: 20-25Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 10Matoltsy A.G. Odland G.F. J. Biophys. Biochem. Cytol. 1955; 1: 191-196Crossref PubMed Scopus (10) Google Scholar, 11Elias P.M. Friend D.S. J. Cell Biol. 1975; 65: 180-191Crossref PubMed Scopus (578) Google Scholar, 12Grayson S. Elias P.M. J. Invest. Dermatol. 1982; 78: 128-135Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 13Matoltsy A.G. Matoltsy M.N. J. Invest. Dermatol. 1966; 46: 127-129Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 14Nemes Z. Marekov L.N. Fésüs L. Steinert P.M. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8402-8407Crossref PubMed Scopus (225) Google Scholar, 15Steinert P.M. Marekov L.N. Mol. Biol. Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (125) Google Scholar, 16Wertz P.W. Swartzendruber D.C. Kitko D.J. Madison K.C. Downing D.T. J. Invest. Dermatol. 1989; 93: 169-172Abstract Full Text PDF PubMed Google Scholar). It is assembled from soluble (e.g. involucrin and small proline-rich proteins) and non-soluble (e.g. loricrin, periplakin, and envoplakin) proteins (17Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. J. Invest. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar, 18Kalinin A.E. Kajava A.V. Steinert P.M. Bioessays. 2002; 24: 789-800Crossref PubMed Scopus (389) Google Scholar). TG1 catalyzes the formation of protein-protein bonds in which the amine acceptor is provided by the ϵ-amino group of a protein-bound lysine and the ultimate link is a N6-(γ-glutamyl)lysine isopeptide bond (19Folk J.E. Annu. Rev. Biochem. 1980; 49: 517-531Crossref PubMed Scopus (872) Google Scholar, 20Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (787) Google Scholar). The cornified envelope is an essential component of the epidermal barrier. Indeed a key role for TG1 in barrier assembly is indicated by impaired barrier function in Tgm1 knock-out mice (21Kuramoto N. Takizawa T. Takizawa T. Matsuki M. Morioka H. Robinson J.M. Yamanishi K. J. Clin. Invest. 2002; 109: 243-250Crossref PubMed Scopus (105) Google Scholar). TG1 function is also required for normal epidermal function in humans. TG1 mutations are present in 50% of autosomal recessive congenital ichthyosis patients. Autosomal recessive congenital ichthyosis is a debilitating skin disease characterized by scaly epidermis and reduced barrier function. Over 90 different mutations of the Tgm1 gene have been identified in these patients (22Farasat S. Wei M.H. Herman M. Liewehr D.J. Steinberg S.M. Bale S.J. Fleckman P. Toro J.R. J. Med. Genet. 2009; 46: 103-111Crossref PubMed Scopus (72) Google Scholar). Many of these mutations are deletion or point mutations within the catalytic domain, but disease-associated mutations are also located in other segments of the TG1 protein that do not include residues that are directly required for activity (22Farasat S. Wei M.H. Herman M. Liewehr D.J. Steinberg S.M. Bale S.J. Fleckman P. Toro J.R. J. Med. Genet. 2009; 46: 103-111Crossref PubMed Scopus (72) Google Scholar, 23Herman M.L. Farasat S. Steinbach P.J. Wei M.H. Toure O. Fleckman P. Blake P. Bale S.J. Toro J.R. Hum. Mutat. 2009; 30: 537-547Crossref PubMed Scopus (69) Google Scholar). These mutations are associated with reduced TG1 level and activity in tissue and cultured cells derived from patients (24Huber M. Yee V.C. Burri N. Vikerfors E. Lavrijsen A.P. Paller A.S. Hohl D. J. Biol. Chem. 1997; 272: 21018-21026Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). transglutaminase type 1 endoplasmic reticulum trimethylamine-N-oxide multiplicity of infection binding immunoglobulin protein fluorescein cadaverine dimethyl sulfoxide enhanced green fluorescent protein empty vector. We know little about how TG1 is trafficked within cells and the impact of disease-associated mutation on these processes. In the present report we study TG1 trafficking and the impact of TG1 mutation on this process and on cell phenotype. Our studies indicate that TG1 is trafficked and processed in the ER and then delivered to the plasma membrane. In contrast, ichthyosis-associated TG1 mutants accumulate in the endoplasmic reticulum and are ubiquitinylated and also shuttled to aggresomes. We propose that inappropriate accumulation of mutant TG1 in intracellular organelles is a potential underlying cause of autosomal recessive congenital ichthyosis. Primary cultures of human foreskin keratinocytes were cultured in 0.09 mm calcium-containing keratinocyte serum-free medium (25Sturniolo M.T. Chandraratna R.A. Eckert R.L. Oncogene. 2005; 24: 2963-2972Crossref PubMed Scopus (33) Google Scholar, 26Sturniolo M.T. Dashti S.R. Deucher A. Rorke E.A. Broome A.M. Chandraratna R.A. Keepers T. Eckert R.L. J. Biol. Chem. 2003; 278: 48066-48073Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For stratified air/liquid interface cultures, two million keratinocytes were plated on a Millicell PCF membrane (0.4 μm, 12-mm insert, 0.6-cm2 surface area) and maintained in Epilife medium supplemented with 1.5 mm calcium for 4 days prior to harvest. Construction of tAd5-TG1-FLAG was previously described (27Jans R. Sturniolo M.T. Eckert R.L. J. Invest. Dermatol. 2008; 128: 517-529Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). tAd5-EV is an empty adenovirus (28Dashti S.R. Efimova T. Eckert R.L. J. Biol. Chem. 2001; 276: 8059-8063Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29Dashti S.R. Efimova T. Eckert R.L. J. Biol. Chem. 2001; 276: 27214-27220Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The tAd5 adenovirus encodes the tetracycline operator element linked to the cytomegalovirus promoter. This promoter is active in the presence of the tetracycline-bound transactivator protein, which is provided by co-infection with an Ad5-transactivator adenovirus (28Dashti S.R. Efimova T. Eckert R.L. J. Biol. Chem. 2001; 276: 8059-8063Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29Dashti S.R. Efimova T. Eckert R.L. J. Biol. Chem. 2001; 276: 27214-27220Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). For experiments, keratinocytes were incubated with 2.5–10 m.o.i. of tAd5-EV or tAd5-TG1-FLAG in the presence of 5 m.o.i. of Ad5-transactivator in keratinocyte serum-free medium containing 6 μg/ml of Polybrene (Sigma). pcDNA3-TG1-FLAG(R142C) and pcDNA3-TG1-FLAG(V379L) were produced by site-directed mutagenesis to convert R142C and V379L. pcDNA3-TG1-FLAG(C377A) and pΔE1Sp1Btet-TG1-FLAG(C377A) were produced by site-directed mutagenesis to convert cysteine 377 of TG1 to alanine. pΔE1Sp1Btet-TG1-FLAG(C377A) was then packaged with the pJM17 adenovirus backbone to produce the tAd5-TG1-FLAG(C377A) adenovirus. PCR primers were used to create the TG1 mutant lacking the N-terminal 52 amino acids and having a FLAG epitope at the amino terminus. The PCR product was cloned into EcoRI/XbaI-restricted pcDNA3 to produce pcDNA3-FLAG-TG1(Δ1–52). For plasmid transfection, 60% confluent keratinocytes were transfected with plasmid using FuGENE 6 (Roche Applied Science) (30Efimova T. Broome A.M. Eckert R.L. J. Biol. Chem. 2003; 278: 34277-34285Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Cells were either fixed for immunofluorescence or lysed for immunoblot analysis 24, 48, or 72 h post-transfection. Immunological detection was performed as described previously (25Sturniolo M.T. Chandraratna R.A. Eckert R.L. Oncogene. 2005; 24: 2963-2972Crossref PubMed Scopus (33) Google Scholar, 26Sturniolo M.T. Dashti S.R. Deucher A. Rorke E.A. Broome A.M. Chandraratna R.A. Keepers T. Eckert R.L. J. Biol. Chem. 2003; 278: 48066-48073Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For immunofluorescence, keratinocytes, grown on coverslips, were infected with adenoviruses, or transfected with plasmids, and at 24, 48, or 72 h post-treatment the cells were washed, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, and methanol permeabilized for 10 min at −20 °C. The coverslips were then incubated for 1 h each with the appropriate antibodies. After washing, the samples were affixed to slides using Mowiol 4-88 (Calbiochem, San Diego, CA), and fluorescence was visualized using a Zeiss LSM510 confocal, or an Olympus OX81 spinning-disc confocal microscope. For immunoprecipitation, total cell extract (150 μg of protein), prepared in lysis buffer, was pre-cleared by addition of 25 μl of protein A/G-agarose for 1 h at 25 °C. The lysate was then combined with 50 μl of lysis buffer-equilibrated anti-FLAG-agarose and incubated overnight at 4 °C. The anti-FLAG-agarose was then washed thoroughly with lysis buffer, boiled, and the entire lysate was loaded on a polyacrylamide gel followed by detection of co-precipitated proteins by immunoblot. Rabbit polyclonal anti-type 1 transglutaminase produced against amino acids 731–817 of human TG1 was from Santa Cruz Biotechnology (sc-25786), rabbit polyclonal anti-FLAG (F7425) was from Sigma, and rabbit polyclonal anti-K14 was obtained from Covance (PRB-155P). Murine monoclonal antibodies include anti-TG1 produced against amino acids 2–33 of human TG1 (sc-166467), anti-ubiquitin (sc-8017), and anti-γ-tubulin (sc-17788) from Santa Cruz; anti-BiP (610798), anti-calnexin (610524), and anti-GM130 (610822) were from BD Transduction Laboratory. Anti-FLAG M2 (F1804), peroxidase-conjugated anti-FLAG M2 (A8592), anti-FLAG M2-agarose (A2220), and anti-β-actin (A5441) were obtained from Sigma. Anti-β-tubulin (ab11311-200) was obtained from Abcam. Alexa-conjugated secondary antibodies were purchased from Molecular Probes (A11008, A11029, A21429, A21424, 11046). Peroxidase-conjugated donkey anti-rabbit IgG (NA934) and peroxidase-conjugated sheep anti-mouse IgG (NA931) were purchased from Amersham Biosciences. Fluorescein cadaverine (FC) (5-((5-aminopentyl)thioreidyl)fluorescein) was obtained from Molecular Probes (A10466). Trimethylamine-N-oxide (TMAO) was purchased from Aldrich (317594). Brefeldin A was obtained from Sigma (B5936). Nocodazole was purchased from Calbiochem (486928). Cells were washed with phosphate-buffered saline, harvested by scraping, and collected by centrifugation. Cell pellets were collected on ice and extracted for 10 min in lysis buffer containing 0.25 m sucrose, 10 mm triethanolamine, 1 mm EDTA, 1 mm β-mercaptoethanol, and protease inhibitor mixture, and passed 30 times through a 27.5-gauge needle. The resulting extract was centrifuged at 1,000 × g three times. The final supernatant was centrifuged at 100,000 × g for 1 h to yield microsomes and cytosol. An equal number of cell equivalents of the cytosol and microsome fraction were boiled and electrophoresed for immunoblot. For microsome extraction experiments, identical aliquots of microsome suspension were divided into four tubes and individual tubes were incubated for 30 min on ice with a final concentration of 1 m NaCl, 0.1 m Na2CO3 (pH 11), or 1% Triton X-100. The samples were centrifuged at 100,000 × g for 1 h and the soluble and insoluble fractions were dissolved at 37 °C by addition of SDS to a final concentration of 4%. For proteinase K challenge assay, microsomes were resuspended in protease inhibitor mixture-free lysis buffer. Individual identical aliquots were incubated with 100 μg/ml of Proteinase K with or without 1% of Triton X-100 on ice for 30 min. Phenylmethylsulfonyl fluoride was then added to the resulting fractions prior to immunoblot. Keratinocytes were infected with appropriate adenovirus and at 48 h fixed in 0.1 m PIPES buffer (pH 7.4) containing 4% paraformaldehyde. The cells were harvested by gentle scraping, washed with phosphate-buffered saline, pelleted, and embedded in 2.5% low melting temperature agarose. Agarose blocks were trimmed to 1 mm3, washed, and dehydrated by progressively lowering the temperature from 4 to −20 °C and increasing the ethanol concentration. The blocks were infiltrated and embedded in unicryl at −20 °C for 48 h. Ultrathin sections were cut using a Leica UltraE microtome (Leica Microsystems, Inc., Bannockburn, IL) and collected on formvar-coated nickel grids. For immunogold labeling, grids were placed section-side down on a drop of phosphate-buffered saline (pH 7.4) containing 1% BSA, 1% fish gelatin, 0.01 m glycine (blocking solution) for 10 min, and then transferred onto a 10-μl droplet of primary antibody (Santa Cruz, sc25786) diluted in blocking solution and incubated for 30 min at 25 °C. The grids were washed five times in 30 ml of phosphate-buffered saline containing 0.1% BSA and 1% fish gelatin. Antibody binding was visualized using 10-nm gold particle-conjugated goat anti-rabbit IgG (British Biocell International) diluted in blocking solution and incubated and washed as for the primary antibody. Grids were then fixed for 5 min with phosphate-buffered saline containing 2% glutaraldehyde, washed with water, and air dried. Samples were visualized using a Tecnai T12 transmission electron microscope at 80 kV, and images were acquired using an AMT digital camera. Transglutaminase catalyzed incorporation of [3H]putrescene into dimethylcasein was previously described (31Kasturi L. Sizemore N. Eckert R.L. Martin K. Rorke E.A. Exp. Cell Res. 1993; 205: 84-90Crossref PubMed Scopus (26) Google Scholar, 32Sizemore N. Kasturi L. Gorodeski G. Eckert R.L. Jetten A.M. Rorke E.A. Differentiation. 1993; 54: 219-225Crossref PubMed Scopus (23) Google Scholar). To monitor activity in cultured cells, keratinocytes, growing on coverslips, were treated with the appropriate adenovirus for 24 h followed by addition of fresh virus-free medium. At 44 h post-infection, fresh keratinocyte serum-free medium, containing 100 μm FC (Molecular Probes, A-10466), was added and the incubation was continued for an additional 4 h (26Sturniolo M.T. Dashti S.R. Deucher A. Rorke E.A. Broome A.M. Chandraratna R.A. Keepers T. Eckert R.L. J. Biol. Chem. 2003; 278: 48066-48073Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To detect disulfide bonds, keratinocytes were infected with tAd5-TG1-FLAG and after 48 h extracts were prepared in phosphate-buffered saline containing 1% Triton X-100. Equivalent quantities of protein were boiled in the presence or absence of dithiothreitol for 5 min and electrophoresed on a 7.5% acrylamide gel lacking reducing agent but containing SDS (33Braakman I. Hebert D.N. Curr. Protoc. Protein Sci. 2001; 14: 1-15PubMed Google Scholar, 34Liscaljet I.M. Kleizen B. Braakman I. Buchner J. Kiefhaber T. Handbood of Protein Folding. Vol. 3. Wiley-VCH Verlag GmbH & Co., Weinheim2004: 73-104Google Scholar) followed by immunoblot with anti-FLAG. TG1 level and activity are low in undifferentiated cultured normal human epidermal keratinocytes grown in serum-free medium (25Sturniolo M.T. Chandraratna R.A. Eckert R.L. Oncogene. 2005; 24: 2963-2972Crossref PubMed Scopus (33) Google Scholar, 26Sturniolo M.T. Dashti S.R. Deucher A. Rorke E.A. Broome A.M. Chandraratna R.A. Keepers T. Eckert R.L. J. Biol. Chem. 2003; 278: 48066-48073Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 35Steinert P.M. Chung S.I. Kim S.Y. Biochem. Biophys. Res. Commun. 1996; 221: 101-106Crossref PubMed Scopus (64) Google Scholar), a finding that is consistent with in vivo studies showing that TG1 is absent in undifferentiated cells in epidermis (36Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (936) Google Scholar, 37Rice R.H. Chakravarty R. Chen J. O'Callahan W. Rubin A.L. Adv. Exp. Med. Biol. 1988; 231: 51-61PubMed Google Scholar, 38Michel S. Démarchez M. J. Invest. Dermatol. 1988; 90: 472-474Abstract Full Text PDF PubMed Google Scholar). Thus, to study TG1 intracellular function we delivered TG1 using a tetracycline-regulated adenovirus, tAd5-TG1-FLAG, that permits expression of full-length TG1-FLAG. Fig. 1A shows that the expressed TG1-FLAG and endogenous TG1 localize to the plasma membrane (arrows), which is consistent with previous observations (39Steinert P.M. Kim S.Y. Chung S.I. Marekov L.N. J. Biol. Chem. 1996; 271: 26242-26250Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 40Phillips M.A. Qin Q. Mehrpouyan M. Rice R.H. Biochemistry. 1993; 32: 11057-11063Crossref PubMed Scopus (45) Google Scholar). This association is confirmed by the observation that TG1-FLAG is present in the particulate (membrane) fraction (Fig. 1B). A requirement for these experiments is expression of TG1-FLAG at physiological levels. To demonstrate this, we compared the TG1 level in tAd5-EV and tAd5-TG1-FLAG virus-infected monolayer keratinocytes with the endogenous TG1 level in stratified keratinocyte cultures grown at the air-liquid interface. Air-liquid interface cultures mimic in vivo keratinocyte differentiation (41Poumay Y. Dupont F. Marcoux S. Leclercq-Smekens M. Hérin M. Coquette A. Arch. Dermatol. Res. 2004; 296: 203-211Crossref PubMed Scopus (145) Google Scholar). The increase observed in raft cultures is consistent with a previous report from Steinert and colleagues (35Steinert P.M. Chung S.I. Kim S.Y. Biochem. Biophys. Res. Commun. 1996; 221: 101-106Crossref PubMed Scopus (64) Google Scholar) that TG1 levels increase more than 100-fold upon keratinocyte differentiation. As shown in Fig. 1C, the TG1 level in tAd5-TG1-FLAG-infected keratinocytes does not exceed the level observed in differentiated air-liquid interface cultures indicating that the expressed TG1 level is in the physiological range. As anticipated, expression of TG1 caused production of cornified envelope-like structures (Fig. 1D, upper panels). These structures display properties of cornified envelopes including resistance to challenge with detergent and reducing agent. Fig. 1D (lower panels) shows survival of cross-linked structures in TG1-FLAG expressing cells after treatment with detergent and reducing agent. To assess activity of expressed TG1-FLAG, particulate and cytosol fractions (Fig. 1B) were assayed for activity. Fig. 1E shows that activity is associated with the particulate fraction and increases with enzyme level. These findings indicate that the expressed TG1-FLAG is active and distributes in the cell in a pattern typical of endogenous TG1. We next assessed the intracellular behavior of several TG1 mutants (Fig. 2A). The TG1 includes membrane-anchoring, β-sandwich, catalytic core, and β-barrel domains (22Farasat S. Wei M.H. Herman M. Liewehr D.J. Steinberg S.M. Bale S.J. Fleckman P. Toro J.R. J. Med. Genet. 2009; 46: 103-111Crossref PubMed Scopus (72) Google Scholar, 42Kim S.Y. Kim I.G. Chung S.I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Abstract Full Text PDF PubMed Google Scholar). Three residues (Cys377, His436, and Asp459) in the TG1 catalytic domain are essential for enzyme activity (43Pedersen L.C. Yee V.C. Bishop P.D. Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). We first studied a catalytically inactive active-site mutant, TG1-FLAG(C377A). As indicated in the confocal z-series (Fig. 2B), we observe perinuclear accumulation of TG1-FLAG(C377A), although some is also detected at the plasma membrane. This differs from the plasma membrane localization of wild-type TG1-FLAG (Fig. 1A). A second mutant, FLAG-TG1(Δ1–52) lacks the N-terminal 52 amino acids that encode a domain that is thought to be essential for membrane association (40Phillips M.A. Qin Q. Mehrpouyan M. Rice R.H. Biochemistry. 1993; 32: 11057-11063Crossref PubMed Scopus (45) Google Scholar). FLAG-TG1(Δ1–52) localizes to the cytoplasm (Fig. 2C). As shown in Fig. 2D, a difference in expression level does not explain the difference in distribution of these three proteins. We next assessed the localization of two additional TG1 mutants. These point mutations (V379L and R142C) are associated with ichthyosis (23Herman M.L. Farasat S. Steinbach P.J. Wei M.H. Toure O. Fleckman P. Blake P. Bale S.J. Toro J.R. Hum. Mutat. 2009; 30: 537-547Crossref PubMed Scopus (69) Google Scholar). Val379 is close to the active site and Arg142 is located upstream in the β-sandwich domain. Keratinocytes were transfected with plasmids encoding TG1-FLAG(V379L) and TG1-FLAG(R142C). As shown in Fig. 2E, these mutants distribute in a perinuclear location similar to that observed for TG1(C377A). In addition, to exclude the possibility that the FLAG epitope has an impact on distribution, we monitored the distribution of the C377A mutant lacking the FLAG sequence. As shown in Fig. 2E, the distribution of TG1(C377A) is identical to that observed for TG1-FLAG(C377A) (Fig. 2B). Little is known regarding the mechanism of TG1 intracellular trafficking except that it is proposed to shuttle between the cytoplasm and plasma membrane dependent upon the presence of myristoyl or palmitoyl lipids attached to the N-terminal anchor domain (35Steinert P.M. Chung S.I. Kim S.Y. Biochem. Biophys. Res. Commun. 1996; 221: 101-106Crossref PubMed Scopus (64) Google Scholar, 44Chakravarty R. Rice R.H. J. Biol. Chem. 1989; 264: 625-629Abstract Full Text PDF PubMed Google Scholar). Because TG1 localizes to the plasma membrane (2Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. J. Invest. Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 45Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. Prog. Exp. Tumor Res. 2005; 38: 115-124Crossref PubMed Scopus (13) Google Scholar), we explored the possibility that it may be trafficked via the ER. Fig. 3A shows that endogenous TG1 and TG1-FLAG distribute to the cell membrane (red, arrows) and, in particular, to sites of cell-cell contact, and that FLAG-TG1(Δ1–52) localizes in the cytoplasm. However, these proteins do not localize with BiP (green), a rough ER protein (Fig. 3A). BiP (binding immunoglobulin protein) is a resident protein of the ER lumen that acts as a chaperone to facilitate correct protein folding (46Ni M. Lee A.S. FEBS Lett. 2007; 581: 3641-3651Crossref PubMed Scopus (650) Google Scholar). It is commonly used as an ER marker. Results presented in Fig. 2B indicate that TG1-FLAG(C377A) localizes at a perinuclear location, suggesting it may be associated with the ER. Consistent with ER localization, we show that TG1-FLAG(C377A) colocalizes with BiP (Fig. 3A, yellow staining and arrow) in 40% of TG1-FLAG(C377A) expressing cells, indicating that ER accumulation is a frequent outcome for this mutant (47Vembar S.S. Brodsky J.L. Nat. Rev. Mol. Cell Biol. 2008; 9: 944-957Crossref PubMed Scopus (1035) Google Scholar). In contrast, we observe perinuclear accumulation in only 3.1% of TG1-FLAG expressing cells (not shown), indicating that wild-type TG1 does not accumulate in the ER. To provide evidence that wild-type TG1-FLAG is trafficked via the ER, we treated cells with brefeldin A, an ER transport blocker (48Nebenführ A. Ritzenthaler C. Robinson D.G. Plant Physiol. 2002; 130: 1102-1108Crossref PubMed Scopus (369) Google Scholar). Brefeldin A treatment results in accumulation of wild-type TG1-FLAG in the ER (Fig. 3A). We also monitored for localization with the cis-Golgi marker, GM130 (49Short B. Barr F.A. Curr. Biol. 2003; 13: R311-R313Abstract Full Text Full Text PDF PubMed Google Scholar). We did not observe colocalization of endogenous TG1, TG1-FLAG, or TG1 mutants with GM130 (Fig. 3C). Moreover, EGFP-galactotransferase, a marker of the trans-Golgi apparatus, does not colocalize with the wild-type or mutant TG1 (not shown). These findings suggest that wild-type TG1 is rapidly trafficked through the ER but that TG1-FLAG(C377A) accumulates in the ER. To further pinpoint subcellular location, TG1-FLAG and TG1-FLAG(C377A) were expressed in keratinocytes and distribution was monitored by immunogold electron microscopy. When expressed at physiological levels TG1-FLAG localizes with the ER in a small percentage (3.1%) of cells and these rare cells cannot readily be located in the EM. When expressed at identical levels, TG1-FLAG(C377A) accumulates in the ER in the majority of cells and causes ER swelling (Fig. 4). Such swelling is not observed when TG1-FLAG is expressed at physiological levels, but is observed when TG1-FLAG is expressed at five times higher levels (Fig. 4). As expected, no signal and no morphological changes were observed in EV-infected cells (Fig. 4). These findings demonstrate that TG1-FLAG(C377A), when expressed at physiological levels, tends to accumulate in the ER, but TG1-FLAG does not. We next used biochemical methods to examine TG1-FLAG and TG1-FLAG(C377A) subcellular distribution. Microsome and cytosol fractions were prepared by 100,000 × g centrifugation. As shown in Fig. 5A, TG1-FLAG and TG1-FLAG(C377A) are enriched in the microsomal (M) fraction, as is the ER marker, calnexin (50Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (465) Google Scholar). To characterize TG1-FLAG membrane association, the microsomal suspension was extracted with 1 m NaCl, which extracts peripheral membrane proteins, or elevated pH (0.1 m Na2CO3, pH 11), which releases intraluminal proteins (51Ohlendieck K. Methods Mol. Biol. 2004; 244: 283-293PubMed Google Scholar). As shown in Fig. 5B, TG1-FLAG is not extracted by high salt (NaCl) or elevated pH (pH 11), but is extracted by treatment with Triton X-100, which extracts membrane proteins. We next assessed whether TG1-FLAG is facing the ER lumen. Proteins that are inside the ER are protected from proteinase K digestion. We show" @default.
• W2040258749 created "2016-06-24" @default.
• W2040258749 creator A5000749676 @default.
• W2040258749 creator A5012791211 @default.
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• W2040258749 creator A5080979590 @default.
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• W2040258749 date "2010-10-01" @default.
• W2040258749 modified "2023-10-12" @default.
• W2040258749 title "Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis" @default.
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