Profilaggrin Requires Both Linker and Filaggrin Peptide
Total Page：16
File Type：pdf, Size：1020Kb
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Profilaggrin Requires both Linker and Filaggrin Peptide Sequences to Form Granules: Implications for Profilaggrin Processing In Vivo1 Melanie K. Kuechle,*† Craig D. Thulin,¶ Richard B. Presland,*† and Beverly A. Dale*†‡§ *Departments of Oral Biology, †Medicine (Dermatology), ‡Periodontics, and §Biochemistry, University of Washington, Seattle, Washington, U.S.A.; ¶Department of Biochemistry, Brigham Young University, Provo, Utah, U.S.A. Filaggrin is an intermediate filament associated protein the linker or conserved phosphorylation sites, nor is that aids the packing of keratin filaments during ter- solubility affected by protein phosphatase 2A treat- minal differentiation of keratinocytes. Premature ment. Granule morphology was abrogated only in a aggregation of keratin filaments is prevented by filag- truncated construct, which still contains the linker. A grin expression as the inactive precursor, profilaggrin, construct consisting of 16 amino acids of filaggrin which is localized in keratohyalin granules in vivo. fused to green fluorescent protein led to rounded and Profilaggrin is phosphorylated and contains multiple bizarrely shaped transfected cells with compact keratin filaggrin repeats separated by a hydrophobic linker filaments, suggesting that very little filaggrin sequence peptide. We have previously shown that filaggrin con- is required for keratin filament interaction. Radio- structs containing the linker, when transiently trans- labeled filaggrin–green fluorescent protein constructs fected into epithelial cells, lead to expression of a specifically bound keratin in overlay assays confirming that the observed cytoskeletal collapse is due to filag- protein that resembles keratohyalin (Dale et al, J Invest grin–keratin interaction. Our findings indicate that Dermatol 108:179–187, 1997). To characterize further profilaggrin must be extensively processed before it the region(s) of the linker and/or filaggrin that are loses both its granule forming ability as well as its necessary for granule formation, we generated several insolubility, suggesting that granule formation in vivo mutant constructs from Flag–FG-1, and generated correlates with insolubility in vitro. Further, filaggrin fusions of filaggrin with green fluorescent protein. We retains its ability to bind keratin as it is degraded to also subjected profilaggrin to protein phosphatase 2A smaller peptides. Key words: filaggrin/green fluorescent treatment and measured its subsequent solubility. We protein fusion protein/keratinization/mutagenesis. J Invest found that granular morphology is not dependent on Dermatol 112:843–852, 1999 eratinization is a term used to describe both the interlocking keratin bundles. This process is rapid, occurring within journey and biochemical changes of a keratinocyte one cell layer, and presumably tightly controlled. as it migrates from the basal proliferative layer to the One identifiable biochemical event at the transition from granular rigid stratum corneum. Keratinization must be a to cornified cell is the conversion of profilaggrin to filaggrin. continuous, orderly, and life-long process to assure the Filaggrin is a hydrophilic, cationic protein that aids aggregation Korganism of an essential barrier between itself and the environment. and subsequent disulfide bonding of keratin filaments (Dale et al, When a component of the process is missing or malfunctioning, 1978). Profilaggrin is the main component of the abundant, epidermal disease ensues (Compton et al, 1992; Dale et al, 1992; basophilic keratohyalin granules from which the granular layer of Epstein, 1992; Fuchs, 1992). The most dramatic and abrupt change the epidermis derives its name. Profilaggrin is a high molecular of keratinization occurs at the granular to stratum corneum transition weight (.400 kDa), phosphorylated protein composed of tandem (Odland, 1991). Here, the keratinocyte must transform itself repeats of filaggrin monomers joined by small linker peptides from a three-dimensional cell complete with active, functioning, (Lonsdale-Eccles et al, 1984; Haydock and Dale, 1986, 1990; intracellular organelles to a flattened, two-dimensional rigid sac of McKinley-Grant et al, 1989). During the transition from granular cell to flattened squame, conversion of profilaggrin to monomeric filaggrin occurs by site-specific proteolysis and dephosphorylation (Haugen-Scofield et al, 1988; Resing et al, 1984, 1989; Kam et al, Manuscript received May 25, 1998; revised December 29, 1998; accepted 1993). The events at the granular to stratum corneum transition for publication February 25, 1999. must coordinate with profilaggrin processing to filaggrin to prevent Reprint requests to: Dr. Melanie Kuechle, Department of Oral Biology/ Medicine (Dermatology), Box 357132, University of Washington, Seattle, premature aggregation of the intermediate filament network. There WA 98185-7132. exist a number of disorders of human epidermis in which the 1This work was presented in part at the Society of Investigative expression and/or processing of profilaggrin to filaggrin is abnormal Dermatology meeting in 1997. (reviewed in Dale et al, 1990), strongly implicating the importance Abbreviations: GFP, green fluorescent protein; PEP1, profilaggrin endo- of timely processing of profilaggrin to filaggrin in normal epidermal protease 1; PP2A, protein phosphatase 2A; REK, rat epidermal keratinocytes homeostasis. 0022-202X/99/$14.00 · Copyright © 1999 by The Society for Investigative Dermatology, Inc. 843 844 KUECHLE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 1. The Flag–FG-1 construct (parental construct) and the four constructs generated from Flag–FG-1. The Flag–1/2Fg construct includes the linker sequence, but terminates immediately after FLY. The Flag–FLY to SRH construct changes the aromatic FLY tripeptide of the linker to SRH, a common tripeptide repeat found in mature filaggrin. The Flag-TS to AA and the Flag-S3 to A3 constructs change conserved phosphorylation sites, which correspond to P1 and P23, respectively (Resing et al, 1995a) to alanines. The parental construct was prepared by cloning from the human profilaggrin gene which contains 11 filaggrin units like FG-1. The arrow indicates the start of the amino terminus of mature filaggrin which contains 126 amino acids plus the 189 amino acids encoded by the following repeat. Expression of human filaggrin constructs containing the linker like sequences, when fused with GFP, however, are able to cause in COS-7 and rat epidermal keratinocytes (REK) leads to granule altered cell shape and cytoskeletal collapse. Radiolabeled filaggrin– formation reminiscent of keratohyalin granules, whereas no granules GFP fusion proteins are able to bind specifically to keratin in an are seen when constructs that do not contain linker are expressed in vitro overlay assay showing that the observed cytoskeletal collapse in these same cell types (Dale et al, 1997). The difference between is due to the keratin-binding ability of the transfected constructs. constructs that lead to granule formation and those that do not is the inclusion of a linker peptide consisting of 10 amino acids, MATERIALS AND METHODS RSGRSGSFLY. The actual length of the linker in vivo is not Preparation of filaggrin and GFP constructs The human filaggrin known, but has been shown to be between three and 15 amino constructs, FG-L and FG 1 L, were prepared using the polymerase chain acids in length (McKinley-Grant et al, 1989; Gan et al, 1991; reaction. Flag–FG-1 and Flag–FG5 were generated by cloning single Thulin and Walsh, 1995), with the aromatic FLY tripeptide as the human filaggrin units into a modified form of pcDNA3 that contains an minimum three. The aromatic nature of FLY is in sharp contrast N-terminal Flag sequence (Dale et al, 1997). Three different mammalian to the markedly cationic composition of the filaggrin repeat. The expression vectors were utilized, two containing the immediate early CMV difference in the composition of the linker and filaggrin sequences promoter (pcDNA3 and pCR3.1, both Invitrogen, Carlsbad, CA) and one suggested that the linker may have a discrete function; perhaps containing the involucrin promoter (Inv (H3700-pL2), gift of Dr. J. Carroll, playing a part in granule formation. Genetics Institute, Andover (MA). The aims of our studies were (i) to clarify the region(s) of Construction of mutated profilaggrin constructs The Flag–1/2Fg construct was filaggrin that are necessary for granule formation, and (ii) to ascertain generated via polymerase chain reaction (PCR) using the oligonucleotides: if granule formation in vivo correlates with the insolubility of 59-CCCGGTACCATGGACTACAAGGACGACGAT-39 (upstream) and profilaggrin in vitro. Considering what is known about profilaggrin 59-CCCCTCGAGGTAGAGGAAAGACCCTGAACG-39 (downstream), processing in conjunction with the findings from the differential and the Flag–FG-1 construct as template (Dale et al, 1997). The oligonucleo- morphology of transiently expressed proteins, we developed three tides contain KpnI and XhoI restriction sites (underlined, upstream and hypotheses to accomplish the first aim. These hypotheses were downstream oligonucleotides, respectively) for cloning. PCR was per- based on the potential function of the aromatic linker peptide by formed for 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C in an AmplitronII Thermolyne thermocycler (Barnstead-Thermolyne, itself, the linker in combination with specific phosphorylation