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

Edit Tarcsa,Eleonora Candi,Tonja Kartasova,William W Idler,Lyuben N Marekov,Peter M Steinert

doi:10.1074/jbc.273.36.23297

Abstract

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 alpha or beta 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 substrate in 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.

Highlights

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 SPR name 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 highly conserved
As expected from its unusually basic pI value, the expressed SPR2 migrated on SDS gels with an apparent molecular mass of about 10 kDa, rather higher than that calculated from its known sequences (6 kDa) (Fig. 1, inset)

Summary

Introduction

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). Many peptides recovered from limited proteolysis experiments of CEs isolated from human foreskin epidermis [32,33,34], cultured epidermal keratinocytes [35, 36],3 and mouse forestomach epithelium [12] 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 [12, 33,34,35,36]. Our new data reveal that, the three enzymes can cross-link SPR2 in vitro, we can conclude that the TGase 3 enzyme is used almost exclusively in epithelia in vivo

Methods

Results

Conclusion