Use of specific glycosidases to probe cellular interactions in the sea urchin embryo

Abstract

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used alpha- and ss-amylase and alpha- and ss-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of alpha-glucosidase and ss-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ss-glucosidase and alpha-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the alpha- and ss-glycosidases.

Fig. 1

SDS-PAGE analysis of glycosidase preparations for possible protease contamination. Panel A: Commercial ß-amylase was incubated with egg albumin (labeled with an “a”) and carbonic anhydrase (labeled with a “b”) as described in Methods. In lane 1, are egg albumin and carbonic anhydrase alone at “0” time; lane 2, egg albumin and carbonic anhydrase plus ß-amylase at “0” time. Lanes 3 and 4; 5 and 6; 7 and 8 are the same combinations of proteins incubated for 1, 6, and 24 h. In the even numbered lanes, the major bands above egg albumin and below carbonic anhydrase are the large and small subunits of ß-amylase, respectively. Panel B: Commercial α-glucosidase (the minor bands g1, g2, and g3) was incubated with egg albumin (labeled as “a”) as described in Methods. Lanes 1 and 2; 3 and 4; 5 and 6; 7 and 8 are duplicate samples incubated for 0, 1, 6, and 24 h, respectively with α-glucosidase.

Fig. 2

Effect of glycosidase treatment on embryonic development: Panel A: control 48 h L. pictus embryos in artificial sea water ASW, pH 8.0. The arrows indicate complete archenterons attached to the blastocoel roof. Panel B: glycosidase-treated 48 h L. pictus embryos which were treated at 24 h postinsemination (the result is the same with either ß-amylase or α-glucosidase treatment). The embryos demonstrate mostly unattached/disorganized archenterons as indicated by the arrows. The scale bar is equal to 60 μm.

Fig. 3

Percentage of L. pictus embryos that demonstrated characteristic archenteron development according to the morphology types shown in the presence of increasing concentrations of α-glucosidase. The data are plotted as mean percentages of morphology type and the complete statistics are presented in Table 1. The legend for each panel is: ◆- embryos with complete archenteron; ■- embryos with disorganized archenteron formation. Panel A: embryos with α-glucosidase in ASW. Panel B: embryos with α-glucosidase in LCASW.

Fig. 4

Percentage of L. pictus embryos that demonstrated characteristic archenteron development according to the morphology types shown in the presence of increasing concentrations of β-amylase. The data are plotted as mean percentages of morphology type and the complete statistics are presented in Table 2. The legend for each panel is: ◆- embryos with complete archenteron; ■- embryos with disorganized archenteron formation; ▲-embryos with no invagination of the vegetal plate; ●-embryos which have exogastrulated. Panel A: embryos with β-amylase in ASW. Panel B: embryos with β-amylase in LCASW.