Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family - PubMed (original) (raw)

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

J A Encinas et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Amino acid sequence alignment of mouse, rat, and human Sema W and genomic structure of mouse Semaw. (Upper) Residues in the rat and human sequence that are identical to the mouse sequence are represented by dots; gaps in sequence relative to the mouse sequence are represented by dashes. The sema domain (solid outline), Ig-like domain (dashed outline), and the transmembrane domain (single underline) are shown. A region of homology to the vaccinia virus protein A39R is indicated by a wavy underline. A subdomain of plexin/SEX homology is indicated by a double underline. The string of leucines encoded by the CTG repeat is indicated by black shading, and a cyclic nucleotide-dependent phosphorylation site is indicated by dark-gray shading. (Lower) The relative positions and sizes of the Semaw exons in the mouse genomic sequence are shown. Exon 1 has been found thus far only in the rat. Intron lengths over 2,000 bp are estimates calculated by comparing intron-spanning PCR products with size markers on an agarose gel.

Figure 2

Figure 2

Northern and in situ hybridization analysis of Sema W expression. Northern blots were performed with a rat Sema W probe against a panel of total RNA extracts from various adult rat tissues (a), various adult rat central nervous system tissues (b), and rat embryonic (E) and postnatal (P) tissues at various stages of development (c; given in days post coitus or days after birth, respectively). Below each Northern blot image, confirmation of equivalent RNA amounts by ethidium bromide staining is shown. Locations of 28S and 18S ribosomal RNA are indicated. Sema W bands are indicated with arrows. (d) In situ hybridization with a rat Sema W probe of a transverse section of an embryonic-day-15 rat spinal column. Darkly staining regions are the motor neurons (M), dorsal root ganglia (DRG), and sympathetic ganglia (SG). (e) In situ hybridization with a rat Sema W probe of a coronal section of an embryonic-day-15 rat eye. Staining is seen in the retina (R) and along the optic nerve tract (ON).

Figure 3

Figure 3

Location of the gene for Sema W in humans and mice. The human SEMAW gene was localized between markers WI-5987 and GCT1B4 by radiation hybrid mapping. The locations of disease loci for Parkinson’s disease and Alström’s syndrome are shown along with the approximate locations of microsatellite markers that define their limits. The relative locations of the markers shown are based on the radiation hybrid map of the human genome mapping project (52). The mouse Semaw gene was localized between markers D6Mit71 and D6Mit9 by radiation hybrid mapping, and nearby markers D6Mit189 and D6Mit70 and the gene Dok were localized by physical mapping. The approximate locations of disease loci of cerebellar deficient folia (cdf), mnd2, and truncate (tc) are shown relative to the mapped markers. cR, centirays.

Figure 4

Figure 4

Expression of Sema W in COS7 cells. Western blotting was performed with an antibody generated against a C-terminal peptide derived from the Semaw sequence. Lanes 1 and 2 are blots of soluble fractions from COS7 cells transfected with a Semaw antisense or sense plasmid, respectively. Lanes 3 and 4 are blots of membrane fractions from COS7 cells transfected with a Semaw antisense or sense plasmid, respectively. A, antisense; S, sense. Molecular mass in kilodaltons is indicated on the left.

Figure 5

Figure 5

Growth-cone collapse activity of Sema W. The percentage of collapsed retinal ganglion-cell growth cones in relation to the concentration of total protein added to the growth medium is shown. White bars, membrane fractions from control mock-transfected cells; gray bars, membrane fractions from rat Sema W-expressing cells; black bars, membrane fractions from rat Sema W (Sema-Ig-TM)-myc-expressing cells. The concentrations of the two Sema W forms relative to the total protein concentrations were approximately equal, as determined by Western analysis with an antibody generated against an N-terminal peptide sequence from Sema W (data not shown). ∗, P < 0.05; ∗∗, P < 0.005, compared with controls.

References

    1. Luo Y, Raible D, Raper J A. Cell. 1993;75:217–227. - PubMed
    1. Messersmith E K, Leonardo E D, Shatz C J, Tessier-Lavigne M, Goodman C S, Kolodkin A L. Neuron. 1995;14:949–959. - PubMed
    1. Kolodkin A L, Matthes D J, O’Connor T P, Patel N H, Admon A, Bentley D, Goodman C S. Neuron. 1992;9:831–845. - PubMed
    1. Yu H H, Araj H H, Ralls S A, Kolodkin A L. Neuron. 1998;20:207–220. - PubMed
    1. Matthes D J, Sink H, Kolodkin A L, Goodman C S. Cell. 1995;81:631–639. - PubMed

MeSH terms

Substances

LinkOut - more resources