Abstract In normal epidermis, the expression of keratins 1 and 10 is associated with the loss of proliferative capacity and the onset of terminal differentiation. Keratins 1 (K1) and 10 (K10) are commonly expressed in the differentiating layer of benign tumors, but are lost during progression from the benign to the malignant state in skin carcinogenesis. Active gene constructs of mouse K1 and K10 were introduced into papilloma and carcinoma cell lines derived from keratinocytes to analyze the consequences of the expression of these keratins on the organization of the endogenous cytoskeletal network and on the mitotic activity of the recipient cells. Exogenous K1 integrated into the preexisting keratin K5/K14 network of both SLC‐1 carcinoma and 308 papilloma cells. The formation of a recombinant cytoskeleton was more restricted for K10 than for K1 and appeared to be related to a requirement for cessation of cell division before K10 could integrate. The integration of exogenous K1 filaments into the endogenous keratin network was compatible with sustained proliferation of SLC‐1 carcinoma cells in vitro. However, the exogenous gene was not expressed in tumor grafts in vivo. In contrast, stable K1 or K10 transfectants could not be selected in 308 cells, suggesting that benign tumor cells expressing suprabasal keratins cannot sustain proliferation. Published 1992 Wiley‐liss. Inc.
By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprI and sprII (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate.
Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no α or β structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models. Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no α or β structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models. A large body of recent amino acid sequencing data has demonstrated that members of the three known classes of small proline-rich (SPR) 1The abbreviations used are: SPR, small proline-rich (also, for example, to indicate the SPR1 protein); CE, cornified cell envelope; HPLC, high pressure liquid chromatography; TGase, transglutaminase; NHEK, normal human epidermal keratinocyte(s) 1The abbreviations used are: SPR, small proline-rich (also, for example, to indicate the SPR1 protein); CE, cornified cell envelope; HPLC, high pressure liquid chromatography; TGase, transglutaminase; NHEK, normal human epidermal keratinocyte(s) proteins serve as constituents of the cornified cell envelope (CE) of stratified squamous epithelia (1Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 2Steinert P.M. Marekov L.N. 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Beninati S. Nigra T.P. Folk J.E. Biochem. J. 1988; 271: 305-308Crossref Scopus (40) Google Scholar). The SPR1 (two members), SPR2 (8–11 members) and SPR3 (one member) are assembled from a common plan (10Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar, 11Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Mol. Cell. Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (68) Google Scholar, 12Marvin K.W. George M.D. Fujimoto W. Saunders N.A. Bernacki S.H. Jetten A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11026-11030Crossref PubMed Scopus (147) Google Scholar, 13Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (185) Google Scholar, 14Kartasova T. Darwiche N. Kohno Y. Koizumi H. Osada S.-I. Huh N.-H. Steinert P.M. Kuroki T. J. Invest. Dermatol. 1996; 106: 294-305Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Their amino (head) and carboxyl (tail) domains are enriched in Gln and Lys residues and consist of sequences that have been conserved between each member of an SPR class but differ between classes. These flank a central domain consisting of a series of Pro-rich peptide repeats of sequences that likewise have been conserved between members of a class but vary between classes. The SPRs become cross-linked to themselves and other CE structural protein constituents by both disulfide bonds and N ε-(γ-glutamyl)lysine or N1, N8-bis(γ-glutamyl)spermidine isopeptide bonds formed by transglutaminases (TGases), resulting in an insoluble macromolecular protein complex ideal for barrier function (6Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (170) Google Scholar, 7Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic press, Inc., New York1993: 107-150Google Scholar, 8Simon M. Leigh I.M. Lane E. Watt F.M. The Keratinocyte Handbook. 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Dermatol. 1996; 106: 647-654Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 28Saunders N.A. Jetten A.M. J. Biol. Chem. 1994; 269: 2016-2022Abstract Full Text PDF PubMed Google Scholar, 29Yaar M. Eller M.S. Bhawan J. Harkness D.D. DiBenedetto P.J. Gilchrest B.A. Exp. Cell Res. 1995; 217: 217-226Crossref PubMed Scopus (35) Google Scholar, 30Tesfaigzi J. Wright P.S. Oreffo V. An G. Wu R. Carlson D.M. Am. J. Respir. Cell Mol. Biol. 1993; 9: 434-440Crossref PubMed Scopus (24) Google Scholar, 31Gilchrest B.A. Garmyn M. Yaar M. Arch. Dermatol. 1994; 130: 82-86Crossref PubMed Scopus (58) Google Scholar). Examination of the way in which the SPRs were cross-linked to protein partners in human and mouse CE preparations revealed several novel features of their properties and functions (1Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 2Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 3Robinson N.A. Lapec S. Welter J.F. Eckert R.L. J. Biol. Chem. 1997; 272: 12035-12046Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 4Steinert P.M. Candi E. Kartasova T. Marekov L.N. J. Struct. Biol. 1998; 122: 76-85Crossref PubMed Scopus (83) Google Scholar, 5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). First, only head and tail domain Gln and Lys residues were used for cross-linking. Indeed, multiple adjacent residues were often used simultaneously on the same protein to form a complex interchain, and perhaps intrachain, cross-linked network (3Robinson N.A. Lapec S. Welter J.F. Eckert R.L. J. Biol. Chem. 1997; 272: 12035-12046Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 4Steinert P.M. Candi E. Kartasova T. Marekov L.N. J. Struct. Biol. 1998; 122: 76-85Crossref PubMed Scopus (83) Google Scholar, 5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Each SPR molecule participated in as many as four cross-links (4Steinert P.M. Candi E. Kartasova T. Marekov L.N. J. Struct. Biol. 1998; 122: 76-85Crossref PubMed Scopus (83) Google Scholar). Second, the SPRs were found to be cross-linked to many protein partners, and indeed, they were consistently found to function as cross-bridgers between themselves or between loricrin, involucrin, etc. (4Steinert P.M. Candi E. Kartasova T. Marekov L.N. J. Struct. Biol. 1998; 122: 76-85Crossref PubMed Scopus (83) Google Scholar, 5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Fourth, as initially suggested from expression studies by earlier investigators, our sequencing data directly revealed that the amounts of SPRs present in the CEs from various tissues varied widely; while human foreskin epidermal keratinocyte CEs contained about 5% SPRs (2Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), the CEs of the mouse forestomach contained about 22% SPRs (5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Further modeling analyses revealed that the amounts of SPRs in the epidermal CEs from trunk, lip, and footpad correlate well with the degree of physical and mechanical trauma to which the tissues are normally subjected (5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Thus, we have proposed that cross-bridging SPRs play an important role as modulators of the biomechanical properties of the CEs and the entire epithelium in which they are expressed (5Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In order to further explore these functions of the SPRs, we need to be able to express large quantities of the proteins for study. Since their cross-bridging role is mediated to a substantial degree through TGase cross-linking, we also need to know which enzymes are responsible for their cross-linking in vivo and how this is done. In an initial study, we described the preparation and some properties of a member of the human SPR2 class, and showed that it is cross-linked almost entirely by the TGase 3 enzyme in vitro and in vivo (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this paper, we have extended this work to the study of a member of the human SPR1 class. We show here that its cross-linking is much more complex, since it requires at least two TGases (3 and 1) operating in a sequential manner using different Gln residues for cross-linking to its partners as seen in vivo. A full-length cDNA clone encoding human SPR1 (clone 15B of Ref. 10Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar) was obtained as a generous gift from C. Backendorf. Following the addition of appropriate linkers, it was inserted into the pET-11a bacterial expression vector (Novagen, Madison, WI) and transformed into the host Escherichia coli B strain BL 21/DE3 (Novagen). Protein expression was induced in the presence or absence ofl-[35S]cysteine (2 μCi/ml) as described previously (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Fresh or previously frozen pellets of bacteria were lysed and dialyzed against several changes of 100 volumes of 25 mm sodium citrate (pH 3.6), 1 mmdithiothreitol, 1 mm EDTA, and a mixture of protease inhibitors as described (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). While most of the bacterial proteins precipitated, the SPR1 protein remained soluble. Purification to homogeneity was achieved using an Amersham Pharmacia Biotech fast protein liquid chromatography system on a 0.5 × 5 cm Mono-S column equilibrated in the citrate buffer with a 0–1.0 mNaCl gradient and was eluted with 0.18 m salt. The fractions were analyzed on 4–20% SDS-polyacrylamide gels (Novex, San Diego, CA) with Coomassie stain or by Western blotting using a polyclonal antibody broadly reactive against both mouse and human SPR1 proteins (14Kartasova T. Darwiche N. Kohno Y. Koizumi H. Osada S.-I. Huh N.-H. Steinert P.M. Kuroki T. J. Invest. Dermatol. 1996; 106: 294-305Abstract Full Text PDF PubMed Scopus (60) Google Scholar). The enhanced chemiluminescence detection was performed with the Super Signal CL-HRP Substrate System (Pierce). Alternatively, the SPR1 proteins were monitored by autoradiography. Mouse primary keratinocytes were grown to confluency in low calcium (0.05 mm) medium. After 6 days, they were transferred to medium containing high Ca2+ (1.4 mm), 10 nm staurosporin (to induce SPR1 expression (34Stanwell C. Denning M.F. Rutberg S.E. Cheng C. Yuspa S.H. Dlugosz A.A. J. Invest. Dermatol. 1996; 106: 482-489Abstract Full Text PDF PubMed Scopus (38) Google Scholar)), 100 μm LTB-2 (transglutaminase inhibitor) (35Killackey J.J.F. Bonaventura B.J. Castelhano A.L. Billedeau R.J. Farmer W. DeYoung L. Krantz A. Pliura D.H. Mol. Pharmacol. 1989; 35: 701-706PubMed Google Scholar) (Syntex Research), and 0.5 μC/ml [35S]cysteine. After 24 h, the cells were harvested in phosphate-buffered saline, lysed by polytron homogenization, and centrifuged (10,000 × g, 10 min, 4 °C). The cytosol was then dialyzed against the citrate buffer used above, under which conditions the bulk of the keratin proteins precipitated, leaving the SPR1a/b proteins highly enriched. Their final purification was done following the method for the bacterial expressed proteins, and the proteins were eluted by 0.2m NaCl. Three TGase enzymes were used. Human full-length TGase 1 was expressed in baculovirus, and the particulate fraction containing the unprocessed full-length but active TGase 1 form was isolated as described (36Candi E. Melino G. Lahm A. Ceci R. Rossi A. Kim I.G. Ciani B. Steinert P.M. J. Biol. Chem. 1998; 273: 13693-13702Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This has a specific activity of about 6 pmol of putrescine incorporation into succinylated casein/h/pmol of TGase 1 protein (36Candi E. Melino G. Lahm A. Ceci R. Rossi A. Kim I.G. Ciani B. Steinert P.M. J. Biol. Chem. 1998; 273: 13693-13702Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), as measured using 14C-putrescine (Amersham Pharmacia Biotech; specific activity, 118 mCi/mmol) and remains fully active for at least 1 day. A typical yield was 5–10 nmol of TGase 1/liter of insect cell culture medium. We also isolated the several TGase 1 isoforms present in the cytosolic and membrane-bound compartments of normal human epidermal keratinocytes (NHEK) grown in high Ca2+ medium in the presence of [35S]methionine for 4 days after reaching confluency, exactly as described previously (37Kim S.-Y. Chung S.-I. Steinert P.M. J. Biol. Chem. 1995; 270: 18026-18035Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 38Steinert P.M. Chung S.-I. Kim S.-Y. Biochem. Biophys. Res. Commun. 1996; 221: 101-106Crossref PubMed Scopus (63) Google Scholar). These included the membrane-bound full-length 106-kDa enzyme with a specific activity of about 5 (as assayed using the above units); the 67/33/10-kDa complex released from membranes by use of Triton X-100 with a specific activity of about 1000; the cytosolic full-length enzyme with a specific activity of about 50; the cytosolic 67/33-kDa complex with a specific activity of about 500; and the cytosolic 67-kDa form with a specific activity of about 250. We have noted that several of these isoforms begin to lose activity at 4 °C within about 6 h of isolation (37Kim S.-Y. Chung S.-I. Steinert P.M. J. Biol. Chem. 1995; 270: 18026-18035Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 38Steinert P.M. Chung S.-I. Kim S.-Y. Biochem. Biophys. Res. Commun. 1996; 221: 101-106Crossref PubMed Scopus (63) Google Scholar), presumably because of unfolding and/or destabilization (36Candi E. Melino G. Lahm A. Ceci R. Rossi A. Kim I.G. Ciani B. Steinert P.M. J. Biol. Chem. 1998; 273: 13693-13702Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The established method of using 14C-iodoacetamide for measurement of TGase 1 molar amounts recovered in each culture experiment takes about 1 day (39Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Abstract Full Text PDF PubMed Google Scholar), and thus to perform experiments using constant molar amounts of enzyme isoforms is problematic because of loss of enzyme activity. However, we have found that when NHEK cells are cultured under consistent conditions, the amounts of [35S]methionine specific activity incorporated into the TGase 1 isoforms (as measured after iodoacetamide titrations) are reproducible and are 21 ± 2 dpm/pmol for the cytosolic 67-kDa form and 40 ± 4 dpm/pmol for all other active forms. (This is to be expected based on the methionine contents (37Kim S.-Y. Chung S.-I. Steinert P.M. J. Biol. Chem. 1995; 270: 18026-18035Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).) Accordingly, we used the amount of 35S label to estimate the molar amount of each TGase 1 isoform recovered from fast protein liquid chromatography columns. In a typical culture of normal human epidermal keratinocytes in a 100-mm dish, we recovered 100–200 pmol of TGase 1, or 15–100 pmol of each of the above membrane-bound or cytosolic isoforms. Guinea Pig Liver TGase 2 Was Obtained Commercially (Sigma). TGase 3 proenzyme, which has no measurable activity, was isolated and purified from guinea pig skin. It was activated by dispase as described previously (40Kim H.-C. Lewis M.S. Gorman J.J. Park S.C. Girard J.E. Folk J.E. Chung S.I. J. Biol. Chem. 1990; 265: 21971-21978Abstract Full Text PDF PubMed Google Scholar). The amount used for reactions was quantitated by amino acid analysis. For in vitrocross-linking studies using the SPR1 proteins as complete TGase substrates and measurement of isodipeptide cross-link formation, the purified SPRs were equilibrated into a buffer of 50 mmTris-HCl (pH 7.5), 50 mm NaCl, 1 mmdithiothreitol, and 1 mm EDTA. Reactions contained 10 μg of 35S-labeled recombinant human or native mouse SPR1 proteins (about 7500 dpm) and about 12.5 pmol of TGase enzyme (∼500 nm enzyme concentration; 250–500 dpm) in a 25-μl volume. Reactions were initiated by the addition of Ca2+ to the mixture (5 mm final concentration) and incubated at 37 °C. Timed aliquots of 1.5 μl each were removed and subjected to total enzymic digestion for measurement of the amount of cross-link formed during the reactions (41Hohl D. Lichti U. Mehrel T. Turner M.L. Roop D.R. Steinert P.M. J. Biol. Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar). In some experiments, the total reactions were analyzed by SDS-polyacrylamide gel electrophoresis on 4–20% gradient gels, blotted onto nitrocellulose membranes, and analyzed by autoradiography. In preparative experiments, 100 μg of unlabeled recombinant human SPR1 were reacted in a volume 250 μl. In this case, we used the baculovirus membrane-bound full-length TGase 1 form and activated TGase 3 at concentrations of 700 nm for 18 h. Experiments showed that the cross-linking had proceeded to completion (see Fig. 3). We also performed a preparative experiment with the high specific activity keratinocyte membrane-bound 67/33/10-kDa isoform at 50 nm enzyme concentration, a reaction that was completed in 2 h. Kinetic constants of the three TGases were determined for the recombinant human SPR1 protein exactly as described before (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,42Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The concentrations and purity of the purified SPR1 proteins and TGase enzymes were determined by amino acid analysis. Uncross-linked or cross-linked SPR1 proteins were digested with trypsin (1:30 by weight; Sigma; sequencing grade) for 6 h at 37 °C, and the peptides were resolved on a Phenomex ODS reverse phase HPLC column (2.1 × 250 mm) containing 0.08% trifluoroacetic acid and with a gradient of 5–65% acetonitrile over 70 min (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The peaks were collected and sequenced on a Porton LF3000 gas phase sequencer as described previously (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 42Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 43Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In the case of the reactions with the TGase 3 enzyme, or TGase 3 followed by TGase 1, peptides were poorly resolved due to extensive cross-linking; in these experiments, 1-min fractions were collected across the peak for sequencing (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Circular dichroism spectra on the intact SPR1 proteins or synthetic peptides (see below) were performed as before (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 43Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The following peptides based on published human SPR1 sequences (13Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (185) Google Scholar) were synthesized and purified by HPLC: human SPR1 head A domain, SSQQQKQPCIPPP; human SPR1 head B domain, PPPQLQQQQVKQPCP; human SPR1 head A + B domain, SSQQQKQPCIPPPQLQQQQVKQPCQ; human SPR1 tail domain, SIVTPPPAQQKTKQK; human SPR1 central domain repeat, (PKVPEPCQ)2, (PKVPEPCQ)4, and (PKVPEPCQ)6. Kinetic constants using the head and tail domain peptides as substrates for putrescine incorporation were determined exactly as described before (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 42Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Recently, we described the preparation of a recombinant human SPR2 protein and demonstrated that it is a favored substrate of the TGase 3 enzyme in vitro and in vivo (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 23297-23303Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In the present study, we have repeated these experiments using recombinant human and native mouse SPR1 proteins. We show here that both the TGase 3 and 1 enzymes are likely to be essential for SPR1 cross-linking in vivo, and in this consecutive order of reaction. Following expression in bacteria using the pET11a system, the human SPR1 protein was enriched from bacterial lysates by dialysis into 25 mm citrate buffer, pH 3.6 (in which it was very soluble), and purified by chromatography on a Mono-S fast protein liquid chromatography column (Fig. 1). The maximal yields were of the order of 2–5 mg/liter, typical of the CE proteins loricrin (33Candi E. Melino G. Mei G. Tarcsa E. Chung S.-I. Marekov L.N. Steinert P.M. J. Biol. Chem. 1995; 270: 26382-26390Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and SPR2 (32Tarcsa E. Candi E. Kartasova T. Idler W.W. Marekov L.N. Steinert P.M. J. Biol. Chem. 199
ABSTRACT The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G2/M phase, which does not require DNA-PKcs.
The expression of SPRR (small proline‐rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C‐terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.
Protein hyper- or hypophosphorylation induced by okadaic acid (OA) treatment was examined using quiescent cultures of the BALB/MK-2 mouse epidermal keratinocytes. Treatment with OA enhanced the phosphorylation of five proteins with molecular weights of 65,000, 55,000, 50,000, 28,000 and 15,000 (p65, p55, p50, p28, and p15, respectively) and decreased that of two proteins with molecular weights of 22,000 and 20,000 (p22 and p20, respectively). The two major phosphorylated proteins, p65 and p55, were identified as type II and type I keratins, respectively, by immunoblotting and immunoprecipitation with keratin specific antibodies. Serine was the only phosphoamino acid residue in hydrolysates of the 32P-labeled keratins purified from OA-treated cells. Two-dimensional tryptic peptide maps of the phosphorylated keratins showed that the hyperphosphorylation was largely due to phosphorylation at several additional sites in both keratins. The hyperphosphorylation of keratins induced by OA treatment resulted in a drastic change in their solubility. This change closely correlated with reorganization of the keratin filament network, which finally collapsed into large perinuclear aggregates. Concomitantly the cells changed from a typical epithelial shape to a round shape. Of several protein kinase inhibitors tested, only staurosporine interfered with this OA-induced morphological change and reorganization of the keratin network.
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