Structural and Transglutaminase Substrate Properties of the Small Proline-rich 2 Family of Cornified Cell Envelope Proteins (1998)

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no α or β structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substratein vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2. The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no α or β structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substratein vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2. small proline-rich, and also to indicate, for example, the SPR2 protein cornified cell envelope high pressure liquid chromatography transglutaminase 4-(2-aminoethyl)benzenesulfonyl fluoride . Small proline-rich (SPR)1 proteins are a group of about 12 closely related proteins that are expressed in terminally differentiating mammalian stratified squamous epithelia (1Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar, 2Kartasova 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, 3Phillips S.B. Kubilus J. Grassi A.M. Goldaber M.L. Baden H.P. Comp. Biochem. Physiol. 1990; 95B: 781-788Google Scholar, 4Gibbs S. Lohman F. Teubel W. van de Putte P. Backendorf C. Nucleic Acids Res. 1990; 18: 4401-4407Crossref PubMed Scopus (60) Google Scholar, 5Marvin 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 (146) Google Scholar, 6Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (184) Google Scholar, 7An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar, 8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 9Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 10Kartasova 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, 11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To date, three classes of SPRs have been identified: SPR1 (two members in all species examined so far), SPR2 (8–11 members), and a single member of the SPR3 class. All are built according to a common plan. They possess (head) amino- and (tail) carboxyl-terminal domains that are enriched in Gln, Lys, and Pro residues of sequences that are characteristic of and highly conserved between members within each class. The SPR name in fact is based on a distinctive central domain consisting of a series of peptide repeats of either eight (SPR1 and SPR3) or nine (SPR2) residues that are enriched in Pro residues and of sequences that vary between the three classes, but members within each class are also highly conserved. The numbers of repeats range from as few as three in all human SPR2 proteins and the smallest mouse SPR2 protein 2T. Kartasova and P. M. Steinert, unpublished data.2T. Kartasova and P. M. Steinert, unpublished data. to more than 23 in SPR3 proteins (6Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (184) Google Scholar, 11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), so that the total protein size ranges from 6 kDa to ≈25 kDa.The expression of SPR proteins varies widely between different epithelia. For example, the epidermis expresses SPR1a (also known as cornifin α) and a limited number of SPR2 members (6Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (184) Google Scholar, 7An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar, 8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 9Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 10Kartasova 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). Other internal epithelia express more abundant amounts of other SPR2 members together with SPR1a.2 The SPR3 (also known as cornifin β) protein is most abundantly expressed in oral epithelia, the esophagus and rodent forestomach epithelium (11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 13Jarnik M. Kartasova T. Steinert P.M. Lichti U. Steven A.C. J. Cell Sci. 1996; 109: 1381-1391Crossref PubMed Google Scholar). Cultured keratinocytes from several epithelial sources tend to express both SPR1a and 1b, as well as a limited selection of other members (5Marvin 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 (146) Google Scholar, 7An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar, 9Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 10Kartasova 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, 11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 14Fujimoto W. Nakanishi G. Arata J. Jetten A.M. J. Invest. Dermatol. 1997; 108: 200-204Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 15Koizumi H. Kartasova T. Tanaka H. Ohkawara A. Kuroki T. Br. J Dermatol. 1996; 134: 686-692Crossref PubMed Scopus (33) Google Scholar, 16An G. Huang T.H. Tesfaigzi J. Garcia-Heras J. Ledbetter D.H. Carlson D.M. Wu R. Am. J. Respir. Cell Mol. Biol. 1992; 7: 104-111Crossref PubMed Scopus (37) Google Scholar, 17An G. Tesfaigzi J. Carlson D.M. Wu R. J. Cell. Physiol. 1993; 157: 562-568Crossref PubMed Scopus (25) Google Scholar, 18Saunders N.A. Smith R.J. Jetten A.M. Biochem. Biophys. Res. Commun. 1993; 197: 46-54Crossref PubMed Scopus (52) Google Scholar, 19Owens D.M. Zainal T.A. Jetten A.M. Smart R.C. J. Invest. Dermatol. 1996; 106: 647-654Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 20Saunders N.A. Jetten A.M. J. Biol. Chem. 1994; 269: 2016-2022Abstract Full Text PDF PubMed Google Scholar). In addition, the amounts expressed vary during differentiation (2–5,8–11, 14–20). Furthermore, a much wider selection of SPR members is expressed in epithelia in response to UV damage (1Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar, 2Kartasova 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, 4Gibbs S. Lohman F. Teubel W. van de Putte P. Backendorf C. Nucleic Acids Res. 1990; 18: 4401-4407Crossref PubMed Scopus (60) Google Scholar,21Yaar 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), chemical treatment (5Marvin 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 (146) Google Scholar, 8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 14Fujimoto W. Nakanishi G. Arata J. Jetten A.M. J. Invest. Dermatol. 1997; 108: 200-204Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 17An G. Tesfaigzi J. Carlson D.M. Wu R. J. Cell. Physiol. 1993; 157: 562-568Crossref PubMed Scopus (25) Google Scholar, 18Saunders N.A. Smith R.J. Jetten A.M. Biochem. Biophys. Res. Commun. 1993; 197: 46-54Crossref PubMed Scopus (52) Google Scholar, 20Saunders N.A. Jetten A.M. J. Biol. Chem. 1994; 269: 2016-2022Abstract Full Text PDF PubMed Google Scholar), and in hyperproliferative (14Fujimoto W. Nakanishi G. Arata J. Jetten A.M. J. Invest. Dermatol. 1997; 108: 200-204Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 15Koizumi H. Kartasova T. Tanaka H. Ohkawara A. Kuroki T. Br. J Dermatol. 1996; 134: 686-692Crossref PubMed Scopus (33) Google Scholar) or malignant (14Fujimoto W. Nakanishi G. Arata J. Jetten A.M. J. Invest. Dermatol. 1997; 108: 200-204Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 19Owens D.M. Zainal T.A. Jetten A.M. Smart R.C. J. Invest. Dermatol. 1996; 106: 647-654Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 22Tesfaigzi 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, 23Gilchrest B.A. Garmyn M. Yaar M. Arch. Dermatol. 1994; 130: 82-86Crossref PubMed Scopus (58) Google Scholar) diseases.Recent experiments have documented that SPR proteins serve as structural protein precursors of the cornified cell envelope (CE), which is a specialized structure that affords essential barrier function to stratified squamous epithelia (24Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (169) Google Scholar, 25Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press, Inc., New York1993: 107-150Google Scholar, 26Simon M. Leigh I.M. Lane E. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar). It is assembled at the cell periphery and constitutes a layer of protein of uniform thickness (about 15 nm) and density (about 7 kDa/nm2) in different epithelia (27Jarnik M. Simon M.N. Steven A.C. J. Cell Sci. 1997; 111: 1051-1060Google Scholar). The CE is made insoluble by extensive cross-linking of several defined structural proteins including the SPRs by disulfide bonds and N ε-(γ-glutamyl)lysine orN 1,N 8-bis(γ-glutamyl)spermidine isopeptide bonds formed by the action of transglutaminases (TGases) (24Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (169) Google Scholar, 25Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press, Inc., New York1993: 107-150Google Scholar, 26Simon M. Leigh I.M. Lane E. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar, 28Martinet N. Beninati S. Nigra T.P. Folk J.E. Biochem. J. 1988; 271: 305-308Crossref Scopus (40) Google Scholar). TGases catalyze the usually irreversible formation of an intra- or more usually intermolecular isopeptide bond between acyl donor Gln and acceptor Lys residues (29Folk J.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar, 30Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Crossref PubMed Scopus (657) Google Scholar, 31Folk J.E. Chung S.-I. Methods Enzymol. 1985; 113: 358-375Crossref PubMed Scopus (250) Google Scholar). Unambiguous evidence that SPRs serve as CE precursors and are in fact good substrates for TGasesin vivo comes from direct sequencing analyses. Many peptides recovered from limited proteolysis experiments of CEs isolated from human foreskin epidermis (32Steinert P.M. Cell Death & Differ. 1995; 2: 23-31PubMed Google Scholar, 33Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 34Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), cultured epidermal keratinocytes (35Robinson 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, 36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar), 3P. M. Steinert and L. N. Marekov, unpublished data.3P. M. Steinert and L. N. Marekov, unpublished data. and mouse forestomach epithelium (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) contained recognizable SPR sequences cross-linked through one or more isopeptide bonds to other CE proteins. Examination of these peptides revealed that Gln and Lys residues on only the head and tail sequences of the SPRs were involved in the cross-links (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 34Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 35Robinson 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, 36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar). Furthermore, the data indicated that the SPRs functioned primarily as cross-bridging proteins, by mediating links between other proteins, often loricrin in the epidermis (33Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 34Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar), or loricrin, trichohyalin, and themselves in the forestomach CEs (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). They also form cross-links between themselves in the CEs of cultured keratinocytes that may serve as a model system for other internal stratified squamous epithelia (36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar).However, little information is available to date on the biophysical and biochemical properties of the SPR proteins. Although individual members of the SPR family can function as both amine donors and acceptors for the TGase 1 enzyme in vitro (8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar), few data are available on which TGase(s) are responsible for cross-linking them to CE structures in epithelia in vivo. Three TGases are commonly expressed in epithelia, including the TGase 1, TGase 2, and TGase 3 enzymes (24Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (169) Google Scholar, 25Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press, Inc., New York1993: 107-150Google Scholar, 26Simon M. Leigh I.M. Lane E. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar). In this paper, we have initiated a detailed analysis of their structures and properties. We have expressed in bacteria a member of the human SPR2 class, examined some of its structural features, and explored its substrate properties by the three TGase enzymes. Our new data reveal that, although the three enzymes can cross-link SPR2in vitro, we can conclude that the TGase 3 enzyme is used almost exclusively in epithelia in vivo.DISCUSSIONAmino acid sequencing analyses have demonstrated previously that members of each of the three known classes of SPR proteins are widely used for the assembly of the CE of stratified squamous epithelia, a specialized structure which is of critical importance for barrier function (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 34Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 35Robinson 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, 36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar). Although those studies have provided a wealth of information on which residues are used for cross-linking by TGasesin vivo, and the potential structural and functional consequences of the observed cross-linking (36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar), it was not clear which enzyme(s) were involved, and whether the enzymes display substrate and or sequence specificities. To begin to address these questions, in this paper we have developed methods for the expression and purification of a member of the human SPR2 class, and have used it to explore in vitro its cross-linking properties by three of the TGases widely expressed in stratified squamous epithelia.We have generated three types of data, which together suggest that the recombinant human SPR2 protein used here had correctly folded following isolation from bacteria. First, by CD analyses, the limited degree of structural order of the proteins could be reversibly recovered on addition and then removal of guanidine hydrochloride or heat treatment (Fig. 2). Second, we observed for the TGase 1 and 3 enzymes a high degree of sequence specificity of Gln and Lys residues employed for cross-linking, which would not have been expected for a denatured or improperly folded protein (40Tarcsa 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 (137) Google Scholar, 47Simon M. Green H. J. Biol. Chem. 1988; 263: 18093-18098Abstract Full Text PDF PubMed Google Scholar). Third, the usage of Gln and Lys residues in the recombinant SPR2 protein observed in vitrowas similar to that observed previously in protein sequencing data of in vivo human and mouse CE samples (Fig. 5). Therefore, the data obtained from the in vitro experiments are likely to be biologically relevant in vivo. In this regard, we have found that several other epidermal structural proteins can be refolded following extraction and purification by use of denaturing or non-physiological solvents, including the keratin intermediate filament chains (48Steinert P.M. Idler W.W. Zimmerman S.B. J. Mol. Biol. 1976; 108: 547-567Crossref PubMed Scopus (240) Google Scholar), filaggrin (49Steinert P.M. Cantieri J.S. Teller D.C. Lonsdale-Eccles J.D. Bale B.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4097-4101Crossref PubMed Scopus (222) Google Scholar), loricrin (39Candi 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 now an SPR2 protein.Our data of Fig. 3 indicate that the recombinant human SPR2 protein was used as a complete substrate by the TGase 1 and 3 enzymes present in the epidermis and other stratified squamous epithelia, but by the TGase 2 enzyme only very weakly, if at all. These data confirm earlier studies which have demonstrated that a variety of SPR proteins of different species serve as TGase substrates in in vitroreactions (5Marvin 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 (146) Google Scholar, 8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Notably, however, we show here that the SPR2 protein was cross-linked most readily by the TGase 3 enzyme in vitro (Fig. 3; Table I), a process that has not been examined by other investigators. Furthermore, we demonstrate in this study that the TGase 1 and 3 enzymes cross-link the SPR2 with significantly different rates and at different residue positions to form interchain oligomeric cross-linked products.We found that all cross-linking in vitro in both the intact recombinant SPR2 protein (Figs. 3 and 4) as well as synthetic peptides (Table I) involved use of Gln and Lys residues located in head and tail domain sequences, in excellent with in (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 34Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 35Robinson 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, 36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar) (Fig. 5). is that the head and tail domains have little or no organized structure (Fig. 2 and those Gln and Lys residues are more to the the Gln and Lys residues of the central domain. The central domain sequences of the SPR2 protein an (Fig. T. Steinert P.M. J. Invest. Dermatol. 1995; 104: Full Text PDF Scopus Google Scholar) which may the residues for the of of Gln and Lys residues of the recombinant SPR2 protein in vitro was similar to that seen in vivo for human foreskin epidermis and cultured human as well as for mouse forestomach epithelium (Fig. 5). For head and tail domain Gln and Lys the were the total of the about could be used by the TGase 1 enzyme, for about of the total Gln residue usage of these data, we may conclude that almost all cross-linking of SPR2 in vivo is by the TGase 3 enzyme, with only a minor degree by TGase about 3 of of SPR2 protein are vivo and in vitro, this that multiple Gln and Lys residues were used on each domain of the in vivo revealed that the SPR2 proteins as interchain between other CE proteins. few may have involved links between head and tail sequences within a single SPR2 as well as interchain links to (36Steinert, P. M., Kartasova, T. & Marekov, L. N. (1998)J. Struct. Biol. in pressGoogle Scholar). the TGase 3 enzyme formed interchain in vitro (Fig. we the that some cross-links between head and tail domain sequences were formed as now be to explore the cross-linking properties of the other members of the SPR with the present data the of SPRs as of the properties of the CEs and epithelia in which are expressed (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Small proline-rich (SPR)1 proteins are a group of about 12 closely related proteins that are expressed in terminally differentiating mammalian stratified squamous epithelia (1Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar, 2Kartasova 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, 3Phillips S.B. Kubilus J. Grassi A.M. Goldaber M.L. Baden H.P. Comp. Biochem. Physiol. 1990; 95B: 781-788Google Scholar, 4Gibbs S. Lohman F. Teubel W. van de Putte P. Backendorf C. Nucleic Acids Res. 1990; 18: 4401-4407Crossref PubMed Scopus (60) Google Scholar, 5Marvin 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 (146) Google Scholar, 6Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (184) Google Scholar, 7An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar, 8Greco M.A. Lorand L. Lane W.S. Baden H.P. Parameswaran N.P. Kvedar J.C. J. Invest. Dermatol. 1995; 104: 204-210Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 9Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 10Kartasova 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, 11Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To date, three classes of SPRs have been identified: SPR1 (two members in all species examined so far), SPR2 (8–11 members), and a single member of the SPR3 class. All are built according to a common plan. They possess (head) amino- and (tail) carboxyl-terminal domains that are enriched in Gln, Lys, and Pro residues of sequences that are characteristic of and highly conserved between members within each class. The SPR name in fact is based on a distinctive central domain consisting of a series of peptide repeats of either eight (SPR1 and SPR3) or nine (SPR2) residues that are enriched in Pro residues and of sequences that vary between the three classes, but members within each class are also highly conserved. The numbers of repeats range from as few as three in all human SPR2 proteins and the smallest mouse SPR2 protein 2T. Kartasova and P. M. Steinert, unpublished data.2T. Kartasova and P. M.