Recombinant Mouse Desmoglein-1-beta (Dsg1b), partial

Introduction

Desmogleins (Dsgs) are transmembrane glycoproteins that belong to the cadherin superfamily of calcium-dependent adhesion molecules . They are core components of desmosomes, which are intercellular adhesion junctions that provide mechanical strength and stable cell-cell contacts to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells . Desmoglein-1 (Dsg1) is a specific member of the desmoglein family that is expressed in the upper epidermis .

Discovery of Dsg1-beta

A novel mouse desmoglein gene, sharing high homology to both mouse and human Dsg1, was cloned . The previously published mouse Dsg1 gene was designated as Dsg1-alpha, and the new gene was named Dsg1-beta . The full-length mouse Dsg1-beta cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids . Dsg1-beta protein shares 94% and 76% identity with mouse Dsg1-alpha and human DSG1, respectively .

Gene Structure and Expression

Analysis of the intron/exon organization of the Dsg1-alpha and Dsg1-beta genes revealed significant conservation . RT-PCR analysis showed that Dsg1-beta mRNA was detected in 17-day-old embryos only, while Dsg1-alpha mRNA was expressed in 15- to 17-day-old embryos and adult spleen and testis .

Subcellular Localization

A FLAG-tagged expression construct of Dsg1-beta was transiently expressed in epithelial HaCaT cells to assess subcellular localization . Dsg1-beta-FLAG was found at the cell-cell border and was recognized by the anti-Dsg1/Dsg2 antibody DG3.10 .

Function of Dsg1

Dsg1 is crucial for maintaining skin integrity and barrier function in layered tissues . It plays a role in cell-to-cell adhesion and junction assembly among skin cells . Dsg1 is involved in maintaining fibrous protein stability, which is essential for skin cell renewal and preventing toxin-induced skin detachment . Dsg1 is also regulated by transcription factors like Klf5, which aids in corneal stability and basal cell proliferation . This protein is associated with skin barrier formation and repair, as well as controlling allergic reactions . Dsg1 is required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program .

5.1. Dsg1 and Cell Adhesion

Dsg1 promotes the formation of intercellular adhesion complexes, and the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process . The ratio of Dsg1 to desmocollin 1 (Dsc1) is a critical determinant of desmosomal adhesion in fibroblasts .

5.2. Dsg1 and Keratinocyte Differentiation

Dsg1 supports keratinocyte differentiation and suprabasal morphogenesis . Dsg1 can promote keratinocyte differentiation even when the N-terminal residues required for adhesion have been removed .

Dsg1 in Disease

Dsg1 can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases . Defects in tissue integrity are believed to result from compromised desmosomal adhesion .

Recombinant Production

Recombinant Mouse Desmoglein-1-alpha (Dsg1a), partial can be produced by co-cloning the gene encoding the mouse Desmoglein-1-alpha (Dsg1a) (50-564aa) with the N-terminal 10xHis-SUMO-tag gene and C-terminal Myc-tag gene into a plasmid vector, leading to the formation of a recombinant plasmid . The resulting recombination plasmid is transformed into E.coli cells . E.coli cells containing the recombinant plasmid survive in the presence of a specific antibiotic and are selected to be cultured under conditions conducive to the expression of the gene of interest . Following expression, the recombinant mouse Dsg1a protein is isolated and purified from the cell lysate using affinity purification . Denaturing SDS-PAGE is utilized to resolve the resulting recombinant mouse Dsg1a protein, demonstrating a purity exceeding 85% .

Immune Response

T-cell clones specifically respond to recombinant Dsg1 (rDsg1) . The immune response of FS T-cell clones to rDsg1 is restricted to HLA-DR molecules .

Table 1: HLA typing result of FS patients