SUMMARY:To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or 10-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes. (Lab Invest 2000, 80:1583-1592.

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.

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