title: 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 site-directed mutagenesis, two distinct roles of the amino- and carboxyl-terminal sequences of SPRR1 have been demonstrated. First, we demonstrated that the amino-terminal 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. Site-directed 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 cross-linked 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.
authors.affiliation: Department of Internal Medicine, University of California at Davis, Davis, California 95616, USA.
authors.forename: J
authors.initials: J
authors.lastname: Deng
authors.forename: R
authors.initials: R
authors.lastname: Pan
authors.forename: R
authors.initials: R
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D001980:Bronchi / Q000737:chemistry*; D002478:Cells, Cultured; D055233:Cornified Envelope Proline-Rich Proteins; D003432:Cross-Linking Reagents / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004848:Epithelium / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014132:Trachea / Q000737:chemistry*
chemical_list: D055233:Cornified Envelope Proline-Rich Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D003513:Cycloheximide