Identification and Characterization of a Novel Secretory Granule Calcium-binding Protein from the Early Branching Eukaryote Giardia lamblia*□S

Giardia lamblia is a flagellate protozoan that infects humans and other mammals and the most frequently isolated intestinal parasite worldwide. Giardia trophozoites undergo essential biological changes to survive outside the intestine of their host by differentiating into infective cysts. Cyst formation, or encystation, is considered one of the most primitive adaptive responses developed by eukaryotes early in evolution and crucial for the transmission of the parasite among susceptible hosts. During this process, proteins that will assemble into the extracellular cyst wall (CWP1 and CWP2) are transported to the cell surface within encystation-specific secretory vesicles (ESVs) by a developmentally regulated secretory pathway. Cyst wall proteins (CWPs) are maintained as a dense material inside the ESVs, but after exocytosis, they form the fibrillar matrix of the cyst wall. Little is known about the molecular mechanisms involved in granule biogenesis and discharge in Giardia, as well as the assembly of the extracellular wall. In this work, we provide evidences that a novel 54-kDa protein that exclusively localizes to the ESVs is induced during encystation similar to CWPs, proteolytically processed during granule maturation, and able to bind calcium in vitro. The gene encoding this molecule predicts a novel protein (called gGSP for G. lamblia Granule-specific Protein) without homology to any other protein reported in public databases. Nevertheless, it possesses characteristics of calcium-sequestering molecules of higher eukaryotes. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this secretory granule protein regulates Ca -dependent degranulation of ESVs during cyst wall formation. The protozoan Giardia lamblia colonizes the upper small intestine of several vertebrate hosts and is one on the most common causes of intestinal disease worldwide. This parasitic organism has a simple life cycle, alternating between the disease-causing trophozoite and the environmentally resistant cyst, which is responsible for the transmission of the disease among susceptible hosts. After the reception of the stimulus that triggers encystation, G. lamblia undergoes several metabolic and morphologic changes. These comprise the synthesis, processing, and transport of leucine-rich repeat-containing proteins that will be finally secreted and assembled at the cell exterior to form a protective cyst wall (1, 2). From their synthesis in the endoplasmic reticulum (ER), cyst wall proteins (CWPs) must be transported to the cell membrane in a very effective and controlled manner because the survival of the parasite outside the intestine of the host depends on the success of this process (3). Unfortunately, the knowledge of the transport machinery in Giardia is limited, mostly due to the fact that trophozoites lack essential secretory organelles, such as morphologically identifiable Golgi apparatus and secretory granules (4, 5). However, constitutive protein secretion in Giardia is exemplified by the continuous transport to the plasma membrane and release into the culture medium of variant-specific surface proteins (VSP) (6, 7), whereas regulated secretion apparently occurs only during encystation (3, 8). The biogenesis of the Golgi complex and of typical secretory granules containing cyst wall materials, called encystationspecific secretory vesicles (ESVs), has been reported to occur at early stages of trophozoite differentiation into cyst (8–10). In higher eukaryotes, proteins that will be secreted are assembled and glycosylated during their transport from the ER, through the Golgi apparatus, to the plasma membrane (11). Regulated secretion of hormones and peptides from endocrine and exocrine cells, as well as other parasitic organisms (12, 13), occurs via a regulated secretory pathway and involves the storage of secretory proteins into secretory granules until an appropriate stimulus triggers the release of the content of the granule to the cell exterior (11, 14–16). Since Giardia derives from the earliest branch of the eukaryotic line of descent (17), the study of the biogenesis of secretory organelles that occurs during differentiation might provide new insights into the minimal machinery required for protein transport by the regulated secretory pathway in eukaryotes (1–3). Moreover, the ability to * This work was supported by the Agencia Nacional para la Promocion de la Ciencia y la Tecnologia (ANPCYT), Fundacion Antorchas, Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AF293411 and AF293409. □S The on-line version of this article (available at http://www.jbc.org) contains three supplementary figures illustrating the purification of ESVs from encysting Giardia trophozoites; Western blot analysis of Giardia trophozoites showing that protein recognized by the mAb is induced during encystation similar to CWPs; and Western blots showing that gGSP is in a soluble form inside secretory vesicles. ¶ To whom correspondence should be addressed: Cátedra de Bioquı́mica y Biologı́a Molecular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Pabellón Argentina 2 piso, Ciudad Universitaria, CP 5000, Córdoba, Argentina. Tel. /Fax: 54-351-433-3024; E-mail: hlujan@biomed.uncor. edu. 1 The abbreviations are: ER, endoplasmic reticulum; CWP, cyst wall protein; mAb, monoclonal antibody; ESV, encystation-specific secretory vesicle; gGSP, G. lamblia granule-specific protein; gsp, G. lamblia granule-specific protein gene; NSF, N-ethylmaleimide-sensitive factor; SNARE, soluble NSF attachment protein receptors; HA, hemagglutinin; nt, nucleotides. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 52, Issue of December 27, pp. 50557–50563, 2002 Printed in U.S.A.