Sirpa Aho

The Protein Core of the Proteoglycan Perlecan Binds Specifically to Fibroblast Growth Factor-7

Perlecan is a multifaceted heparan sulfate proteoglycan that is expressed not only as an intrinsic constituent of basement membranes but also as a cell-surface and pericellular proteoglycan. Perlecan functions as a ligand reservoir for various growth factors that become stabilized against misfolding or proteolysis and acts as a co-receptor for basic fibroblast growth factor by augmenting high affinity binding and receptor activation. These biological properties are mediated by the heparan sulfate moiety. Rather little is known about the protein cores mediation of functions. We have recently discovered that fibroblast growth factor-7 (FGF7) binds to perlecan protein core and that exogenous perlecan efficiently reconstitutes FGF7 mitogenic activity in perlecan-deficient cells. In this report we examined the specific binding of FGF7 to various domains and subdomains of perlecan protein core. Using several experimental approaches including overlay protein assays, radioligand binding experiments, and the yeast two-hybrid system, we demonstrate that FGF7 binds specifically to the N-terminal half of domain III and to a lesser extent to domain V, with affinity constants in the range of 60 nm. Thus, perlecan protein core should be considered a novel biological ligand for FGF7, an interaction that could influence cancer growth and tissue remodeling.

Unique role for the periplakin tail in intermediate filament association: specific binding to keratin 8 and vimentin

Abstract: Plectin, desmoplakin, and the 230‐kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue‐wide survey utilizing RT‐PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C‐terminal domains. The short C‐terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two‐hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co‐expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co‐localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well‐characterized plakin proteins plectin and desmoplakin, and thus may function tissue‐wide as a scaffolding protein in intermediate filament assembly.

Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high‐affinity protein/protein interactions

Abstract:  Extracellular matrix protein 1 (ECM1), an approximately 85‐kDa glycoprotein with broad tissue distribution, harbors mutations in lipoid proteinosis (LP), a heritable disease characterized by reduplication of basement membranes and hyalinization of dermis, associated with neurologic disorders. The mechanisms leading from ECM1 mutations to LP phenotype are unknown. In this study, we explored ECM1 protein‐protein interactions utilizing yeast two‐hybrid genetic screen of human placental library, which identified nine interacting proteins, including matrix metalloproteinase 9 (MMP9). The interactions were confirmed by β‐galactosidase assay with isolated clones and by co‐immunoprecipitation which narrowed the interacting segment in ECM1 to the C‐terminal tandem repeat 2 (amino acids 236–361). This peptide segment also inhibited MMP9 activity in a gelatin‐based ELISA assay. We propose that ECM1‐mediated reduction in MMP9 proteolytic activity may have relevance to pathogenesis of LP.

180‐kD bullous pemphigoid antigen/type XVII collagen: Tissue‐specific expression and molecular interactions with keratin 18

The 180‐kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane protein, also known as type XVII collagen. In this study, potential interactions of BPAG2 with other proteins expressed in epidermal keratinocytes were explored by yeast two‐hybrid system using the amino‐terminal intracellular domain of BPAG2 as a bait. Several independent interacting clones encoding keratin 18 (K18) were identified when the keratinocyte cDNA library, cloned into the yeast two‐hybrid activation domain vector, was screened. The peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15–25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site‐directed mutagenesis abolished the interaction. Further examination of the K18 sequences by restricted cDNA constructs in yeast two‐hybrid system identified a carboxyl‐terminal segment corresponding to helix 2B domain as critical for BPAG2 binding. The interaction of BPAG2/K18 was confirmed by an in vitro protein‐protein interaction assay, which also confirmed that normal human keratinocytes express K18 in culture. The tissue specific expression of BPAG2 was first examined using a multi‐tissue RNA blot. Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR‐templates to study the expression patterns of both BPAG2 and K18. The results demonstrated significant level of expression of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with predominant epithelial component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma. As expected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has a relatively broad tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have direct interactions with K18, a keratin characteristically expressed in a simple epithelia. J. Cell. Biochem. 72:356–367, 1999.

A new family of polymorphic genes in Saccharomyces cerevisiae: α-galactosidase genes MEL1-MEL7

SummaryUsing genetic hybridization analysis we identified seven polymorphic genes for the fermentation of melibiose in different Mel+ strains of Saccharomyces cerevisiae. Four laboratory strains (1453-3A, 303-49, N2, C.B.11) contained only the MEL1 gene and a wild strain (VKM Y-1830) had only the MEL2 gene. Another wild strain (CBS 4411) contained five genes: MEL3, MEL4, MEL5, MEL6 and MEL7. MEL3-MEL7 were isolated and identified by backcrosses with Mel− parents (X2180-1A, S288C). A cloned MEL1 gene was used as a probe to investigate the physical structure and chromosomal location of the MEL gene family and to check the segregation of MEL genes from CBS 4411 in six complete tetrads. Restriction and Southern hybridization analyses showed that all seven genes are physically very similar. By electrokaryotyping we found that all seven genes are located on different chromosomes MEL1 on chromosome II as shown previously by Vollrath et al. (1988), MEL2 on VII, MEL3 on XVI, MEL4 on XI, MEL5 on IV, MEL6 on XIII, and MEL7 on VI. Molecular analysis of the segregation of MEL genes from strain CBS 4411 gave results identical to those from the genetic analyses. The homology in the physical structure of this MEL gene family suggests that the MEL loci have evolved by transposition of an ancestral gene to specific locations within the genome.

Plakin proteins are coordinately cleaved during apoptosis but preferentially through the action of different caspases

Abstract:  In epithelial cells, cell–cell and cell–matrix junctions, desmosomes and hemidesmosomes, provide anchorage sites for the keratin‐intermediate filaments. The plakin proteins desmoplakin (DP), plectin, and periplakin represent intracellular constituents of these adhesion junctions. In staurosporine‐treated apoptotic HaCaT cells, DP, plectin, and periplakin became cleaved coordinately with the elimination of keratins 10 and 14, while involucrin, actin, and keratin 18 displayed considerable stability. The caspase inhibitor zVAD‐fmk prevented both the cell detachment and protein cleavage, indicating the function of caspases in these events. Closer examination in vitro revealed that while caspases 2 and 4 most efficiently cleaved DP, and plectin served as a target for caspases 3 and 7, periplakin as well as keratins were cleaved by caspase 6. The involvement of multiple caspases in the destruction of epithelial cell integrity ensures the efficient elimination of cytoskeleton, but also provides specificity for selectively targeting individual adhesion molecules.

Periplakin Gene Targeting Reveals a Constituent of the Cornified Cell Envelope Dispensable for Normal Mouse Development

ABSTRACT The members of the plakin family of proteins serve as epidermal cytolinkers and components of cell-cell and cell-matrix adhesion complexes, i.e., desmosomes and hemidesmosomes, respectively. Periplakin is a recently characterized member of this family. Human and mouse periplakin genomic loci are conserved, and the proteins are highly homologous, suggesting a role for periplakin in vertebrate physiology. In order to evaluate the functional role of periplakin, we generated periplakin null mice through targeted homologous recombination of mouse embryonic stem cells, followed by development of Ppl−/− mice. Mice homozygous for the targeted allele were born in the expected Mendelian frequency, developed normally, possessed grossly normal epidermis and hair, and were healthy and fertile. The epidermal barrier appeared to develop normally during fetal days E15.5 to E16.5, and the cornified envelope and desmosomes in the newborn mice were ultrastructurally normal. No compensatory increase in the expression of other epithelial proteins was detected in the neonatal mouse epidermis lacking periplakin. Consequently, the primary role of periplakin may not relate to the physiology of the cornified cell envelope in epidermal keratinocytes but may reside in the challenges, which normal laboratory mice do not encounter.

Soluble form of Jagged1: Unique product of epithelial keratinocytes and a regulator of keratinocyte differentiation

Notch signaling pathway is an important regulator of epithelial differentiation. Recent studies have characterized multiple ligands, including Jagged1, as mediators of Notch signaling. In this work, an alternatively spliced transcript of Jagged1 was isolated in yeast two‐hybrid screening through interaction with thrombospondin‐1. This transcript, devoid of sequences encoding the transmembrane and intracellular domains of Jagged1, was specific for keratinocytes. Furthermore, the truncated Jagged1 polypeptide devoid of the intracellular domain was detected prominently in the suprabasal keratinocyte layer in neonatal epithelia. The soluble form of Jagged1, when expressed as a tagged polypeptide was efficiently secreted into the culture medium and the N‐terminal signal sequence became cleaved off upon secretion. Direct contact between a cell presenting the transmembrane form of Jagged1 and a Notch‐expressing cell has been proposed as a prerequisite for the signaling event. However, the soluble form of Jagged1, when present in the cell culture medium, was sufficient to induce keratinocyte differentiation. These observations suggest a novel mechanism of how Jagged1 can target cells beyond the direct cell–cell contact in developing epithelia.

Tissue Distribution and Cell Type-specific Expression of p120ctn Isoforms

Cadherin-based molecular complexes play a major role in cell-cell adhesion. At the adherens junctions the intracellular domain of cadherins specifically interacts with β-catenin and p120ctn, members of the Armadillo repeat protein family. Differential splicing and utilization of the alternative translation initiation codons lead to many p120ctn isoforms. Two major p120ctn isoforms are expressed in mouse tissues. In this study we used indirect immunofluorescence to demonstrate significant tissue specificity in expression of the p120ctn isoforms. The short isoform is abundant at cell-cell adhesion junctions in epidermis, palatal, and tongue epithelia, in the ducts of excretory glands, bronchiolar epithelium, and in mucosal epithelia of esophagus, forestomach, and small intestine. In contrast, the long isoform, containing an amino terminus highly conserved within the p120ctn subfamily, is expressed at vascular-endothelial cell junctions in blood vessels, at cell-cell junctions in the serosal epithelium lining the internal organs, in choroid plexus of brain, in the pigment epithelium of retina, and in structures such as the outer limiting membrane of retina and intercalated discs of cardiomyocytes. The tissue- and cell type-specific expression of p120ctn isoforms suggests a role for the long p120ctn isoform in cell structures responsible for stable tissue integrity, compared to the role of the short isoform in cell-cell adhesion in the external epithelia with rapid turnover.

Human p120ctn catenin: Tissue‐specific expression of isoforms and molecular interactions with BP180/type XVII collagen

Catenins, a family of structurally related proteins, are involved in epidermal keratinocyte cell‐cell adhesion by interacting through their central Armadillo repeats with the intracellular domains of cadherins, transmembrane components of the adhesion junctions. p120ctn is a catenin expressed in different isoforms due to alternative splicing and multiple translation start sites. BP180 is a collagenous transmembrane protein (type XVII collagen) localized to hemidesmosomal attachment complexes in basal keratinocytes. In this study, we have delineated the molecular interaction between these two proteins utilizing the yeast two‐hybrid system, which was confirmed by an in vitro protein‐protein interaction assay. Specifically, it was shown that an amino‐terminal segment of BP180 (aa. 13–25) contains the information necessary for binding to p120ctn isoforms 1–3, but not to the isoform 4, suggesting that the interacting domain is located immediately upstream from the Armadillo repeats and is encoded by exons 5 and 6, which are subject to alternative splicing only in a minority of transcripts. In addition to epidermal keratinocytes, p120ctn was shown to be expressed in a variety of adult and fetal tissues as well as in a number of human tumors. The expression pattern of various p120ctn transcripts, reflecting alternative splicing of the 5′ exons, was strikingly similar between the corresponding adult and fetal tissues, while the expression patterns were discordant between certain tumors and their normal parental tissues, suggesting a functional role for the tissue‐specific expression of the p120ctn isoforms. Finally, the tissue‐specific expression of BP180 was shown to partially overlap with that of p120ctn, suggesting that the interaction of these two proteins may contribute to the modulation of cell‐cell/matrix interactions in such tissues. J. Cell. Biochem. 73:390–399, 1999.