Spatio-temporal expression of pamlin during early embryogenesis in sea urchin and importance of N-linked glycosylation for the glycoprotein function

Abstract

Expression of pamlin, a heterotrimeric primary mesenchyme cell (PMC) adhesion glycoprotein, and its role during early embryogenesis were examined using immunochemistry and microinjection of pamlin to tunicamycin-treated embryos of the sea urchin, Hemicentrotus pulcherrimus. Pamlin faintly detected in egg cortex before fertilization was strongly expressed in the hyaline layer after fertilization. The embryonic apical surface retained pamlin throughout early embryogenesis, whereas pamlin on the basal surface showed a dynamic change of spatio-temporal distribution from morula to gastrula stage. Pamlin distributed on the entire basal surface of the ectoderm before onset of invagination gradually disappeared from the presumptive archenteron during gastrulation, and then was restricted to the apical tuft region and the PMC sessile sites in early gastrulae. Tunicamycin, an inhibitor of N-glycosydically linked carbohydrate formation, inhibited PMC migration and gastrulation. Tunicamycin also inhibited the assembly of mannose moieties of 180 and 52 kDa subunits of pamlin. Pamlin microinjection to the tunicamycin-treated embryos rescued them from this morphogenetic disturbance. PMCs did not bind to pamlin isolated from the tunicamycin-treated embryos. The present study indicated that pamlin plays an essential role in PMC migration, its termination and gastrulation, and the presence of N-glycosydically linked carbohydrate moieties that contain mannose are necessary to preserve the biological function of pamlin.

Disappearance of an epithelial cell surface‐specific glycoprotein (Epith‐1) associated with epithelial–mesenchymal conversion in sea urchin embryogenesis

“…hardwicki were fixed for 30 min with – 20°C ethanol, respectively. The samples were embedded in Polywax after being dehydrated and infiltrated as reported by Katow and Komazaki (1996), and were then sectioned at 6 μm. After dewaxing by passing three times (each 15 min) through pure ethanol, the samples were air dried and blocked with 3% bovine serum albumin (BSA) in 0.1 M phosphate‐buffered saline (PBS; 39 m M NaH 2 PO 4 •2H 2 O, 61 m M Na 2 HPO 4 •12H 2 O, pH 7.0) for 30 min.…”

“…Hybridoma culture medium conditioned with anti‐Epith‐1 mAb and VIIC7 (a mAb for pamlin that does not bind to any cell surface molecule) (Katow & Komazaki 1996) was condensed 30× using Centriprep 10, and the resultant IgG was purified with a HiTrap Protein G affinity column (Amersham Pharmacia Biotech, Uppsala, Sweden), respectively. The quantity of each IgG was estimated at A 280 , and added (concentration of 50 μg/mL) to the embryonic cells.…”

An RGDS peptide‐binding receptor, FR‐1R, localizes to the basal side of the ectoderm and to primary mesenchyme cells in sand dollar embryos

“…This interpretation could include the involvement of another ECM ligands, such as pamlin (Katow 1995;Katow & Komazaki 1996). These changes are apparently closely related to morphogenetic movement, such as PMC migration in early gastrulae.…”

“…Similarly disturbed morphogenesis occurs by introducing antihuman fibronectin antibodies to the blastocoele (Katow 1990). Subsequently, we have isolated pamlin from sea urchin embryos and shown in vitro that PMC binding to pamlin is severely inhibited in the presence of RGDS peptides (Katow 1995;Katow & Komazaki 1996). Subsequently, we have isolated pamlin from sea urchin embryos and shown in vitro that PMC binding to pamlin is severely inhibited in the presence of RGDS peptides (Katow 1995;Katow & Komazaki 1996).…”

Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos

“…Six-micrometer thick sections were examined as described previously by Katow and Komazaki (27). Briefly, dewaxed Polywax sections were blocked with 5% BSA in 0.1 M PBS for 1 h, incubated with anti-Epith-2 mAb diluted 1:100 in PBS for 1 h, and washed with 0.1 M PBS three times (10 min each).…”