Shi X et al. (2001), Cell surface beta-1,4-galactosyltransferase-I a...

XB-ART-9606

Development 2001 Mar 01;1285:645-54. doi: 10.1242/dev.128.5.645.

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Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling.

Shi X , Amindari S , Paruchuru K , Skalla D , Burkin H , Shur BD , Miller DJ .

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ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

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Species referenced: Xenopus laevis
Genes referenced: sgta zp2 zp3 zp3.2

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	Fig. 1. Schematic diagram of murine long (LGT), short (SGT), mutant long GalTase (∆LGT), and epitope-tagged versions. All three forms of GalTase have the same catalytic domain, stem region and transmembrane domain (TM). Short GalTase is translated from a downstream translation initiation site and is identical to long GalTase, except its N terminus is truncated. Mutant long GalTase is identical to long GalTase except two arginine residues (in bold) in the cytoplasmic domain were mutated to alanine. AU1 and AU5 epitope tags were added to the luminal/extracellular catalytic domain.
	Fig. 2. Xenopus oocytes injected with murine GalTase mRNA expressed GalTase activity at levels approximately 200-times higher than the endogenous GalTase activity in water-injected control eggs. Data shown represent the means ± s.e.m. (triplicate determinations of five representative experiments). An asterisk above the bars indicates a significant difference from the control group (P<0.01).
	Fig. 3. Immunofluorescence localization of GalTase expressed in Xenopus eggs. AU1 epitope-tagged LGT-, SGT- and ∆LGT- expressing oocytes were fixed and stained with AU1 monoclonal antibody and Texas Red-conjugated secondary antibody. Images were obtained by confocal microscopy. The animal pole showed the greatest staining and all the sections presented are through the animal pole. The insets show a section through the animal pole of the entire egg. The bar in A represents 10 μm. (A) Eggs expressing AU1- tagged LGT showed that long GalTase was localized primarily to the plasma membrane. (B) Eggs expressing AU1-tagged SGT demonstrated that short GalTase is located in the egg cytoplasm. (C) Eggs expressing AU1-tagged ∆LGT showed that the mutant GalTase is localized to the plasma membrane, like LGT. (D) AU1 antibody did not bind to eggs that were injected with water as a control. Similar results were obtained using the AU5 tag and AU5 antibody. In controls, the irrelevant antibody was used and no fluorescence was detected.
	Fig. 4. ZP3, but not ZP2, bound to Xenopus oocytes expressing GalTase. Increasing concentrations of each zona glycoprotein were added to Xenopus oocytes expressing GalTase and water-injected control eggs. With increasing zona protein concentration, ZP3 binding increased to saturation, but ZP2 binding did not differ between RNA-injected and water-injected eggs. In this ZP3 preparation, the KD was approximately 9 nM but affinity was variable between ZP3 preparations.
	Fig. 5. Changes observed as Xenopus eggs were activated. (A) Control egg injected with water and treated with GalTase antibodies. Eggs were sectioned, and cortical granules stained with PAS are evident just under the plasma membrane, indicated with an arrow. (B) GalTase-expressing egg treated with GalTase antibody showing release of cortical granules. The arrow shows the exocytosis of cortical granules. The vitelline envelope has separated from the plasma membrane (arrowhead). (C) Eggs expressing GalTase prior to addition of GalTase antibody. (D) The same eggs as in C 10 minutes after addition of GalTase antibody showed contraction of the pigmented zone of the animal pole. (E) GalTase-expressing egg treated with GalTase antibody illustrating the elevation of the vitelline envelope, shown by the arrow.
	Fig. 6. GalTase agonists activated Xenopus eggs expressing long GalTase. (A) Addition of total zona pellucida (ZP) glycoproteins, ZP3, or GalTase antibodies (Imm) to eggs expressing GalTase triggered cortical granule exocytosis, cortical contraction and vitelline envelope elevation. Ligands were added to GalTase- expressing eggs and signs of activation were observed for 20 minutes. GalTase-expressing eggs treated with preimmune antibodies (PI), monovalent Fab fragments (Imm Fab) or ZP2 had background activation rates. Pertussis toxin (PTx) pretreatment of eggs prevented activation. (B) Eggs injected with water had low activation rates. (C) Eggs expressing the short GalTase protein (SGT) or (D) expressing long GalTase with a mutation in the putative G protein binding domain (∆LGT) did not activate in response to agonists. Results in each panel are the means ± s.e.m. (from 3-10 independent experiments using 20-30 eggs per experiment). An asterisk above the bars indicates a significant difference from the groups without an asterisk (P<0.01).
	Fig. 7. GalTase agonists increase GTPγ[35S] binding to membranes from Xenopus oocytes expressing long GalTase. (A) Addition of total zona pellucida (ZP) glycoproteins, ZP3 or GalTase antibodies (Imm) to eggs expressing GalTase increased GTPγ[35S] binding. Addition of preimmune antibodies (PI), monovalent Fab fragments (Imm Fab) or ZP2 activated only background levels of GTPγ[35S] binding. Pertussis toxin (PTx) pretreatment of eggs prevented any change in GTPγ[35S] binding in response to GalTase agonists. (B) Membranes from water- injected control eggs showed no change in GTPγ[35S] binding. (C) Eggs expressing the short GalTase protein or (D) eggs expressing long GalTase with a mutation in the putative G protein binding domain did not respond to GalTase agonists. Data represent the means ± s.e.m. of four independent experiments performed in triplicate. An asterisk above the bars indicates a significant difference from the groups without an asterisk (P<0.05).
	Fig. 8. Addition of ZP3 or GalTase antibodies (Imm) to eggs expressing GalTase increased GTP hydrolysis. Preimmune antibodies (PI) or ZP2 stimulated only background levels of GTPase activity (left panel). Control eggs injected with water did not respond to GalTase agonists (right panel). Data represent means±s.e.m. of four independent experiments. An asterisk above the bars indicates a significant difference from the groups without an asterisk (P<0.05).