Characterisation of monoclonal antibodies against synapsin I

Abstract

The synapsins are a family of four closely related neuronal phosphoproteins localised at the presynaptic nerve terminal [for review see I]. They link small synaptic vesicles to a number of cytoskeletal proteins including actin, tubulin, f o h n and calmodulin. Functions include the regulation of efficiency of neurotransmitter release and they are also implicated in synapse formation. Thus the synapsins are involved in both neurotransmitter exocytosis and neuronal development. In this study, we have prepared a panel of monoclonal antibodies to purified ovine brain synapsin I with a view to using them as probes of synapsin I bindmg functions. Balb/c mice were immunised sub-cutaneously with 10-60 pg at monthly intervals. The protein was emulsified in Freund's complete adjuvant for the first injection and with incomplete adjuvant thereafter. Three days before fusion, the mice were injected intraperitonealy with 60 pg of synapsin I in phosphate buffered saline.Splenocytes were fused with Sp 210 -Ag14 essentially as described previously [2]. The resultant heterokaryotic cells were initially cultured in a HAT medium (RPMI 1640 based medium containing hypoxanthine / aminopterin / thymidme (HAT) and 10 ' YO foetal bovine serum (Gibco)). Initial screening was by enzyme linked immunosorbent assays (ELISAs) on whole synapsin I or the N-terminal head region of the protein produced by collagenase digestion [3]. A large number of positive hybridomas were cloned three times by limited dilution and screened via ELISA. Four highly active antibodies were further screened by Western blotting on whole synapsin I, collagenase-derived head region, or fragments generated by 2-nitro-5-thobenzoic acid (NTCB) cleavage at cysteine residues [4] as shown in figure 1 . Antibodes SNHI and D7EH recognised epitopes contained in the N-terminal portion of the head. SNTl and ClBT were reactive to the C-terminal 'tail' fragments of synapsin I prepared as above (see table 1). No antibodies specific to the 15 kDa mid-synapsin fragment were detected. SNHI, SNTl and D7EH were monospecific for synapsin I and C l B T cross-reacted with five other proteins in an ovine brain homogenate. Immunohistochemistry using SNTl on rat brain sections demonstrated a distinctive pattern of staining predicted for a protein localised at presynaptic nerve terminals. The antibodies also specifically recognised synapsin I in immunoblots of homogenates of rat, cow, rabbit, guinea pig, mouse and human brain. Table 1. Monoclonal antibodies against synapsin 1 Epitope Antibody Ig Class Collagenase fragment NTCB fragments (48 kDa, N-terminal) (31/35 m a ) SNTI IgG I + ClBT IgM + SNHl IgG I + D7EH IeG 1 -L

Ca2+-Dependent Interaction with Calmodulin Is Conserved in the Synapsin Family: Identification of a High-Affinity Site

“…The reaction of two anti-synapsin I monoclonal antibodies with recombinant synapsin IIb was also tested. SNH1 is a monoclonal antibody that recognizes an epitope in the first 50 amino acids of sheep synapsin I (12). The SNH1 epitope lies in the common 'head' region of all mammalian synapsins.…”

“…Monoclonal antibodies SNH1 and SNT1 were used in Western blotting as previously described (12). Synthetic peptides were prepared by J. Hardy (University of Kent Protein Science Facility) using a Shimadzu peptide synthesizer.…”