Synaptotagmins (Syts) are transmembrane proteins with two Ca2+-binding C2 domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca2+ sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379–388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290–18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca2+ concentrations ([Ca2+]) below 10 μM, whereas other isoforms require 200–500 μM [Ca2+] or show no Ca2+-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594–599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1–5 μM [Ca2+] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93–104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C2A domain of Syt VII inhibit Ca2+-triggered secretion of β-hexosaminidase and surface translocation of Lgp120, whereas the C2A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C2A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca2+-dependent lysosome exocytosis.

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII–deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.

OBJECTIVE-Zinc transporter eight (SLC30A8) is a major target of autoimmunity in human type 1A diabetes and is implicated in type 2 diabetes in genome-wide association studies. The type 2 diabetes nonsynonymous single nucleotide polymorphism (SNP) affecting aa 325 lies within the region of highest ZnT8 autoantibody (ZnT8A) binding, prompting an investigation of its relationship to type 1 diabetes.RESEARCH DESIGN AND METHODS-ZnT8A radioimmunoprecipitation assays were performed in 421 new-onset type 1 diabetic Caucasians using COOH-terminal constructs incorporating the known human aa 325 variants (Trp, Arg, and Gln). Genotypes were determined by PCR-based SNP analysis.RESULTS-Sera from 224 subjects (53%) were reactive to Arg 325 probes, from 185 (44%) to Trp 325 probes, and from 142 (34%) to Gln 325 probes. Sixty subjects reacted only with Arg 325 constructs, 31 with Trp 325 only, and 1 with Gln 325 only. The restriction to either Arg 325 or Trp 325 corresponded with inheritance of the respective C-or T-alleles. A strong gene dosage effect was also evident because both Arg-and Trp-restricted ZnT8As were less prevalent in heterozygous than homozygous individuals. The SLC30A8 SNP allele frequency (75% C and 25% T) varied little with age of type 1 diabetes onset or the presence of other autoantibodies.

The intracellular protozoan parasite Trypanosoma cruzi causes Chagas' disease, which affects millions of people in Latin America. T. cruzi enters a large number of cell types by an unusual mechanism that involves Ca2+-triggered fusion of lysosomes with the plasma membrane. Here we show that synaptotagmin VII (Syt VII), a ubiquitously expressed synaptotagmin isoform that regulates exocytosis of lysosomes, is localized on the membranes of intracellular vacuoles containing T. cruzi. Antibodies against the C2A domain of Syt VII or recombinant peptides including this domain inhibit cell entry by T. cruzi, but not by Toxoplasma gondii or Salmonella typhimurium. The C2A domains of other ubiquitously expressed synaptotagmin isoforms have no effect on T. cruzi invasion, and mutation of critical residues on Syt VII C2A abolish its inhibitory activity. These findings indicate that T. cruzi exploits the Syt VII–dependent, Ca2+-regulated lysosomal exocytic pathway for invading host cells.

The Ca2+ sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca2+-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII+/+ controls, Syt VII−/− animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII−/− mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII+/+ or Syt VII−/− CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.