Identification of exocytotic membrane proteins in three rotifer species

Abstract

We confirmed the presence and expression of exocytotic membrane proteins: Syntaxin-1, Syntaxin-4, SNAP-23, and SNAP-25 in the rotifers Brachionus calyciflorus, Lecane quadridentata, and Philodina roseola. These proteins were identified by immunohistochemistry and immunoblot analysis using antibodies against Syntaxin-1, Syntaxin-4, SNAP-23, and SNAP-25. The presence of these proteins were observed mainly in regions of the nervous, reproductive, and glandular systems of these rotifer species, which underlines the fundamental role of exocytotic docking and fusion membrane proteins in the vesicular release of secretory proteins. The immunoblot analysis confirmed the expression of Syntaxin-1, Syntaxin-4, and SNAP-23 in the three rotifer studied, but not of SNAP-25. This study contributes to rotifer biology by revealing the presence of conservative exocytotic machinery such as SNAREs proteins in the phylum Rotifera, including members of the Class Bdelloidea the highest taxa of metazoans where sexual reproduction does not exist.

Reduced expression of exocytotic proteins caused by anti-cholinesterase pesticides in Brachionus calyciflorus (Rotifera: Monogononta)

“…Electrophoresis was performed in SDS-PAGE (13.5%) according to the Protocol of the Laemmli buffer system (Laemmli, 1970) with slight modifications (Pérez- Legaspi et al, 2008). The stacking gel contains upper buffer (0.5 M Trizma-base, 0.4% SDS at pH 6.8), and resolving gel contains lower buffer (1.5 M Trizma-base, 0.4% SDS at pH 8.8).…”

“…Gel was run in a vertical mini-protean system (Bio-Rad, Hercules, CA, USA). After electrophoresis, proteins obtained from polyacrylamide gel were electrotransfered to a PVDF (polyvinyldene difluoride) membrane (Sigma, St. Louis, MO, USA), according to the protocol of Towbin et al (1979) with slight modifications (Pérez- Legaspi et al, 2008). The western blot was performed by blocking the PVDF membranes in solutions composed of TBS milk 5% (0.5 M NaCl, 20 mM Tris-HCl pH 7.5) for 1 hour at room temperature and incubated overnight at 4° C with rabbit polyclonal primary antibodies (Sigma, St. Louis, MO, U.S.A.) such as anti-Syntaxin-1 (1:1000), anti-Syntaxin-4 (1:500), and anti-SNAP-23 (1:500) in blocking buffer .…”

“…In this invertebrates, SNARE proteins Syntaxin-1, Syntaxin-4, SNAP-23 and SNAP-25 have been reported in glandular, reproductive, and nervous systems (Pérez-Legaspi et al, 2008). Therefore, vesicular secretions of various molecules such as chemical messengers are conserved from invertebrates to vertebrates (Burgoyne and Morgan, 2003).…”

“…In rotifers, the secretion and intracellular traffic of neurotransmitters, hormones and other substances of neural, endocrine and exocrin cells are carried through the fusion of both presynaptic plasma membrane and vesicular membrane regulated by the SNARE complex (Gerst, 1999;Pérez-Legaspi et al, 2008). In this invertebrates, SNARE proteins Syntaxin-1, Syntaxin-4, SNAP-23 and SNAP-25 have been reported in glandular, reproductive, and nervous systems (Pérez-Legaspi et al, 2008).…”

Immunodetection of Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Prolactin (PRL) in Brachionus calyciflorus (Rotifera: Monogononta)

“…The cholinergic system is involved in the reproduction and the fertility control because some neuropeptides control the synthesis of selected glycoproteins like the luteinizing hormone (LH) and gonadotropin (Vijayan 1985). The presence of exocitotic proteins such as Sintaxin-1, Sintaxin-4, SNAP-23 and SNAP-25 in rotifers suggests a mechanism of neurosecretion via exocitotic vesicles of enzymes, and other molecules, like hormones, related to glandular secretions (Pérez-Legaspi et al 2008) which are employed in diverse mechanisms. Snell & Joaquim-Justo (2007), suggest that since rotifers are particularly sensitive to androgenic and anti-androgenic substances, their use as models to study the nature and the impact of substances that are potential endocrine disruptors.…”