Distinct Roles for Amino- and Carboxyl-terminal Sequences of SPRR1 Protein in the Formation of Cross-linked Envelopes of Conducting Airway Epithelial Cells

Abstract

The small proline-rich protein, SPRR1, is a marker gene whose expression in conducting airway epithelium is elevated under a variety of conditions that enhance squamous differentiation. The purpose of this study is to elucidate the nature of the SPRR1 sequence involved in cross-linked envelope formation in a tissue/cell type, such as conducting airway epithelium, that normally does not express squamous function except after injury or maintenance in culture. For this, a Flag-SPRR1 fusion protein expression system has been developed. Using the liposome-mediated gene transfer technique on passage 1 culture of human tracheobronchial epithelial (TBE) cells, the Flag-SPRR1 fusion protein can be expressed and detected immunologically by both anti-Flag and anti-SPRR1 antibodies. The incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes can be demonstrated when transfected human passage 1 TBE cultures are treated with phorbol 12-myristate 13-acetate and high calcium (1.5 mM). By deletion and sitedirected mutagenesis, two distinct roles of the aminoand carboxyl-terminal sequences of SPRR1 have been demonstrated. First, we demonstrated that the aminoterminal sequence of SPRR1 protein is required for the incorporation of the fusion protein into cross-linked envelopes, whereas a deletion on the carboxyl-terminal region or on the middle repetitive unit has no effect. Interestingly, insertion of a 24-amino acid peptide of monkey MUC2 repetitive sequence in the amino-terminus of SPRR1 protein had a stimulatory effect. Sitedirected mutagenesis on the following amino acid residues, Lys 7 , Gln 88 , and Lys 89 , which were found previously to participate in the cross-linked envelope formation of keratinocytes, had no detrimental effect on the incorporation. However, mutations on Gln clusters, such as Gln 4 -Gln 6 and Gln 22 -Gln 25 , had detrimental effects on the incorporation. These results suggest an amino-terminal sequence-dependent and multiple crosslinked sites for the incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes of cultured human TBE cells. Second, we demonstrated that the carboxyl terminus of SPRR1 protein is required for a high level of Flag-fusion protein expression. A deletion in the carboxyl region or a mutation on the last lysine residue of the carboxyl end had a detrimental effect on the level of Flag-SPRR1 fusion protein expressed in transfected cells. In contrast, there was only a slight decrease in the level of expression if the amino-terminus was deleted. Interestingly, the efficiency for fusion protein to incorporate into cross-linked envelopes was elevated by the mutation at the carboxyl end. These results suggest distinct roles, perhaps coordinately, for both amino-and carboxyl-terminal sequences in the regulation of the life cycle of SPRR1 protein in cultured TBE cells.

Differentially expressed late constituents of the epidermal cornified envelope

“…LEP members contain structural similarities to known cornified envelope precursors such as SPRR1 and loricrin, including characteristic N-terminal glutamine residues which are major sites for TGase-mediated cross-linking in SPRRs (10,15,26). This similarity strongly suggests that LEP members are TGase substrates.…”

“…The LEP N terminus resembles that of all of the above proteins and contains glutamine residues, identified in loricrin and SPRRs, as key sites for TGase activity (26)(27)(28)(29). LEPs contain proline͞lysine͞cysteine (PKC) repeats in the N-terminal half, a feature of SPRRs, followed by serine͞glycine͞cysteine-rich regions (SGC), characteristic of loricrin.…”

Global Gene Expression Profiles During Acute Pathogen-Induced Pulmonary Inflammation Reveal Divergent Roles for Th1 and Th2 Responses in Tissue Repair

“…Squamous epithelial cell proliferation and differentiation are indicated by the presence of small proline-rich proteins (SPRRs), which form the cornified envelope underlying the plasma membrane of epithelial cells. In fact, squamous differentiation of the tracheobronchial epithelial cells is only seen during injury and repair, suggesting the presence of these processes (38). Although the SPRR1 family has been observed in the lung, the SPRR2 family has not.…”

JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells

“…However, it is well established that SPRRs act as cross-bridging proteins that participate in a cross-linking process of several CCE precursor proteins, such as loricrin (constituting 50-90% of CCE proteins) and involucrin (4,24). It is worth noting that a recent study demonstrated the actual participation of SPRR1B in the cornification of proximal TBE cells (5). Therefore, we speculate that expression of SPRRs is an essential feature of SCD of distal bronchiolar cells and plays a very important role in providing a physical and chemical barrier against the environment.…”