A third member of the synapsin gene family

Abstract

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12-13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Figure 1

Comparison of the primary sequence of human synapsins Ia, IIa, and IIIa. Sequences of human synapsins Ia, IIa, and IIIa are aligned, and identical residues present in all three isoforms are boxed. Domains A, C, and E are shaded.

Figure 2

Domain model of the synapsin family. Domains are schematically represented, drawn to scale, and indicated by A–J. The length of the polypeptide chains is shown at the top in number of residues.

Figure 3

Northern blot analyses using a synapsin IIIa probe. Human Northern blots (CLONTECH) were hybridized to a probe derived from the 3′ UTR of human synapsin IIIa mRNA. Results using a control actin probe are shown at the bottom of each blot. Molecular sizes are shown in kilobases (kb). Arrowheads point to the 13.0-, 7.9-, 3.5-, and 2.0-kb transcripts. (A) Distribution of synapsin IIIa mRNA in various human tissues. The actin probe recognizes α- and β-actin in heart and skeletal muscle but only β-actin in the other tissues. (B) Distribution of synapsin IIIa mRNA in human brain. The sample designated “whole brain” was derived from a different individual than the other samples on these blots.

Figure 4

Distribution of synapsin IIIa protein. Immunoblot analyses of mouse cortex (WT, wild type; 1KO, synapsin I knock-out; 2KO, synapsin II knock-out; DKO, synapsin I/II double knock-out), rat cortex (Rat), and human cortex (Hum) are shown. (A and B) Protein extracts (extract) from WT (50 μg) and DKO (200 μg) brain were loaded in the first two lanes in A or from WT in the first lane B to follow the migration of synapsins Ia, IIa, and IIIa, as indicated by the arrowheads. For immunoprecipitation, the indicated affinity-purified antibody (IP) was incubated with 500 μg of DKO brain protein extract. Antibody–antigen complexes were isolated by using protein A-Sepharose, and the samples were analyzed by immunoblot with the indicated antibody (blot). (C) Expression of synapsins in mouse, rat, and human brain. Protein extracts (50 μg) were loaded in each lane and blots were probed with G304. (D) Expression of synapsins in various mouse tissues. Protein extracts (100 μg) of various tissues were loaded in each lane, and immunoblots were probed with either G304 (Upper) or RU316 (Lower).

Figure 5

Distribution of synapsins Ia, IIa, and IIIa in subcellular fractions of rat forebrain. Autoradiogram of an immunoblot of protein extract (5 μg) from the indicated subcellular fractions that was probed with G304, followed by ¹²⁵I-labeled anti-rabbit antibody (Fab fragments). H, homogenate; S1, postnuclear supernatant; S2, supernatant of P2; P2, crude synaptosomes; LP1, crude synaptic plasma membranes; LS1, supernatant of LP1; LP2, crude synaptic vesicles; LS2, supernatant of LP2 (synaptosol); SG2, synaptic vesicles; SG4, small synaptic membranes; CPG-US, controlled-pore glass purified, untreated synaptic vesicles; CPG-ST, controlled-pore glass purified, salt-treated synaptic vesicles.

Figure 6

Structure of the human synapsin III gene. Alignment of the exons of the human synapsin III gene with the bacterial artificial chromosomes bk415G2, the P1 artificial chromosome dJ309I22, and cosmids, N104C7, N80H12, E86D10, and N28H9, along chromosome 22. Breaks in the structure represent gaps in the nucleotide sequence. As described in the text, bk766E1 overlaps with dJ309I22. The origin of the sequences (with EMBL accession numbers in parentheses) used to align these exons are as follows: bk415G2 (Z83846) and bk766E1 originated from a previously described human library (45); dJ309I22 (Z98256) from a library constructed at the Roswell Park Cancer Institute by the group of Pieter de Jong (http://bacpac.med.buffalo.edu/); and N104C7 (Z82246), N80H12 (Z80902), E86D10 (Z82181), and N28H9 (Z71183) were derived from human chromosome 22-specific cosmid libraries constructed at the Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550. The 10-kb scale marker refers to exons in N80H12, E86D10, and N28H9 only.

Figure 7

Contribution of individual exons to the coding sequence of human synapsin IIIa. The predicted protein sequence encoded by each exon was aligned with the amino acid sequence for synapsin IIIa (residue numbers given at top), and mapped against the corresponding domain structure for this protein.

Figure 8

Location of synapsin III on human chromosome 22. The location of synapsin III on the long arm of chromosome 22 is depicted in relationship to anonymous markers that map to 22q12.2 to 22q13.1. The distances between the markers are indicated in centimorgans.