Novel Reticular Calcium Binding Protein Is Purified on Taipoxin Columns

Abstract

We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute‐phase proteins. Here we report the identification of the second protein as a 49‐kDa lumenal calcium binding protein that we have named taipoxin‐associated calcium binding protein 49 (TCBP‐49). This protein contains six EF‐hand putative calcium binding domains and the carboxyl‐terminal sequence His‐Asp‐Glu‐Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP‐49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP‐49 will allow direct tests of whether TCBP‐49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.

Ca 2+ -binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca 2+ -dependent activation

“…-overlay assay performed in a protein blot format (12,13). This technique is especially useful for identification of proteins containing EF hand Ca 2ϩ -binding sites such as calmodulin, troponin C, TCBP-49, and calexcitin (12,13,18,19). It has also been used to identify putative Ca 2ϩ binding sites in NH 2 -and COOH-terminal domains of Ca 2ϩ -ATPase (13,20) and Ca 2ϩ -release channel͞ryanodine receptor (21).…”

Neuronal Pentraxins Mediate Synaptic Refinement in the Developing Visual System

“…Still, the molecular mechanisms underlying these cellular processes remain incompletely understood (for review, see Cohen-Corey, 2002). The neuronal pentraxins (NPs), NP1 and NP2 (Noland et al, 1994;Reid and Blobel, 1994;Hsu and Perin, 1995;Tsui et al, 1996), and NP receptor (NPR) (Dodds et al, 1997) were identified as synaptic proteins that bind to affinity columns of the snake venom toxin, taipoxin (that presynaptically blocks neurotransmission), and the lumenal calcium-binding protein TCBP49 (Dodds et al, 1995). NP1, NP2, and NPR are 50% identical to each other, and their C-terminal halves are 20-30% identical to classic immune system pentraxins such as the acute phase proteins, C-reactive protein (Whitehead et al, 1990), serum amyloid P protein (Dowton and McGrew, 1990), and the long pentraxin, PTX3 (Breviario et al, 1992).…”

“…NP2 (also called Narp) is dramatically upregulated by neuronal activity (Tsui et al, 1996) and can induce clustering of AMPA receptors when coexpressed in heterologous cells (O'Brien et al, 1999;Xu et al, 2003). On the basis of these properties and their homology to short pentraxins in the innate immune system that mark cells for phagocytosis and degradation by opsonization, we tested the hypothesis that NPs are required for synapse refinement and elimination in the developing CNS (Dodds et al, 1995(Dodds et al, , 1997.…”

Snake Phospholipase A2 Neurotoxins Enter Neurons, Bind Specifically to Mitochondria, and Open Their Transition Pores

“…Fluorophore-conjugated ammodytoxin A (a 14-kDa PLA 2 neurotoxin isolated from the venom of Vipera ammodytes) was detected in the nucleus of hippocampal neurons (14) and in the cytosol of undifferentiated NSC34 cells (15), a mouse neuroblastoma ϫ spinal cord hybrid cell line (16). In addition, Tpx was reported to bind an endoplasmic reticulum-located protein in vitro (17), and ammodytoxin A was found to bind a variety of cytosolic proteins (18,19) and R25, an integral protein of mitochondria (20). As SPANs require Ca 2ϩ for their hydrolytic activity, the biological relevance of these findings was considered to be questionable.…”