A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization - PubMed (original) (raw)

Comparative Study

A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization

M Koch et al. J Cell Biol. 2000.

Abstract

The netrins are a family of laminin-related molecules. Here, we characterize a new member of the family, beta-netrin. beta-Netrin is homologous to the NH(2) terminus of laminin chain short arms; it contains a laminin-like domain VI and 3.5 laminin EGF repeats and a netrin C domain. Unlike other netrins, this new netrin is more related to the laminin beta chains, thus, its name beta-netrin. An initial analysis of the tissue distribution revealed that kidney, heart, ovary, retina, and the olfactory bulb were tissues of high expression. We have expressed the molecule in a eukaryotic cell expression system and made antibodies to the expressed product. Both in situ hybridization and immunohistochemistry were used to describe the cellular source of beta-netrin and where beta-netrin is deposited. beta-Netrin is a basement membrane component; it is present in the basement membranes of the vasculature, kidney, and ovaries. In addition, beta-netrin is expressed in a limited set of fiber tracts within the brain, including the lateral olfactory tract and the vomeronasal nerve. Functional studies were performed and show that beta-netrin promotes neurite elongation from olfactory bulb explants. Together, these data suggest that beta-netrin is important in neural, kidney, and vascular development.

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Figures

Figure 1

Comparison of the predicted full-length amino acid sequences of human (h) and mouse (m) β-netrin. Amino acids in the human sequence that are conserved in mouse are indicated by dashes. A 19–amino acid signal sequence precedes the β-netrin NH2 terminus; an arrowhead marks the signal peptide cleavage site. The predicted amino acid sequence of β-netrin contains three domains (large boxes): an NH2-terminal domain with homology to domain VI of laminin β chains, a domain with homology to the V domain of laminin β chains, and a COOH-terminal domain with homology to the C domain of netrins. Potential glycosylation sites are doubly underlined; all are conserved between human and mouse. EMBL/Genbank/DDBJ accession numbers for the sequences reported here are as follows: mouse β-netrin, AF281278; human β-netrin, AF278532.

Figure 2

Genetic relationship of the netrins with the NH2-terminal regions of selected laminin chains in mouse. Netrin-1 and -3 are closely related to the laminin γ chains, whereas β-netrin shares greater amino acid identity with the laminin β chains.

Figure 3

Transmission electron microscopic images of rβ-N and rβ-NΔC obtained following rotary shadowing. Representative fields are shown. 44% of the images of full-length β-netrin (A and B) appear as dimers, whereas most of those of β-netrin lacking the C domain (rβ-NΔC) appear as monomers (C and D). The dimers appear to associate through an interaction of the V and C domains. The measured average sizes of the images of the dimers are shown in the models adjacent to B and D (n = 30 monomers; 30 dimers).

Figure 4

Electrophoretic profiles of β-netrin expression products and Western analysis using antibodies mAb61, mAb9F11, and pAbR33. (A) Comparison of the electrophoretic mobilities of purified full-length recombinant mouse β-netrin including the His tag (rβ-N+His), full-length β-netrin with the His tag removed (rβ-N), truncated β-netrin including the His tag (rβ-NΔC+His), and recombinant human laminin β2 chain domains III–VI including the His tag (rβ2 +His). M r, relative molecular mass standards. Proteins were visualized with Coomassie brilliant blue R-250. (B) Western blots of recombinant proteins using an anti-His antibody. Aliquots of culture media from 293-EBNA cells expressing full-length or truncated β-netrin were electrophoretically separated and His-containing proteins were detected using an anti-His antibody. Despite the fact that the netrins are minor components of the applied media, only single bands were detected in the positions expected for full-length or truncated β-netrin. Purified full-length β-netrin is detected only before removal of the His tag, indicating that this tag is fully eliminated by cleavage. Purified recombinant laminin β2 chain short arm is present at sufficient concentration to be easily detected by Western blot. The nomenclature for the individual recombinant proteins is as in A. (C) Characterization of anti-β-netrin antibodies by Western analysis of recombinant proteins. Three membrane blots identical to that described in B were probed with the antibodies mAb61, mAb9F11, and pAbR33. The patterns obtained using all three antibodies is identical: all recognize both the full-length and truncated forms of β-netrin, indicating that the epitopes recognized by the mAbs are within domains V or VI, and the polyclonal antiserum also recognizes epitopes within the same domain. None of the antibodies cross-reacts with the laminin β2 chain short arm, nor with its His tag. The nomenclature for the individual recombinant proteins is as in A.

Figure 5

β-Netrin RNA is expressed widely in human and mouse tissues. The same cDNA probe was used to probe adult human tissue blots (A) and a dot blot (B). Only a single major RNA species is detected on the tissue blots. Strong signals are present in spleen, prostate, ovary, heart, kidney and pancreas, mammary gland, and uterus. Only weak signals are present in brain tissue. (C) RT-PCR confirms strong expression of β-netrin RNA in adult mouse kidney, heart, and ovary, as expected from the human blot analyses. In addition, β-netrin RNA is readily detected in several CNS structures including, olfactory bulb and retina. Some expression is observed in other brain regions; however, spinal cord, brain stem, and dorsal root ganglia contain only low levels of β-netrin RNA.

Figure 6

β-Netrin is expressed and deposited in basement membranes throughout the adult rat kidney. β-Netrin was detected by indirect immunofluorescence using three different anti–β-netrin antibodies (indicated). β-Netrin immunoreactivity can be observed within the major and minor arteries (arrows in A), and in the afferent arterioles (B–D). All portions of the tubules are recognized by the antibodies (A–C), as is the mesangium of the glomeruli (B–D). In situ localization of β-netrin RNA shows reaction product deposited throughout the kidney (D; control probe shown in E), consistent with the protein localization. Bars: (A) 100 μm; (B, C, E and F) 50 μm; (D) 25 μM.

Figure 7

β-Netrin is expressed and deposited in the basement membranes of the adult rat fallopian tube and ovary. (A) R33 antiserum shows β-netrin deposition in the basement membranes underlying the fallopian epithelium, and the arterial smooth muscle in the underlying lamina propria. Some reactivity also appears at the epithelial apical surface. (B) In situ hybridization in the ovary demonstrating that secondary follicles have a high level of expression of β-netrin transcripts. Surrounding primary follicles are less well labeled. The scale here is twice C. (C) β-Netrin is expressed and deposited in the basement membranes of the adult rat ovary. With the R33 antiserum, a strong signal is observed in the basement membrane of the secondary follicles, as well as surrounding arterial smooth muscle. The marker is 100 μm. (D) Higher power view of the in situ hybridization in maturing follicles; high levels of expression of β-netrin transcripts are observed in the perifollicular cells. Primary follicles, which surround the maturing ones, are less well labeled. The scale is as in B. (E) β-netrin immunoreactivity in the adult heart. Immunoreactivity is concentrated in the interstitial spaces surrounding cardiac myocytes. The scale is one quarter that of F. (F) In situ hybridization in fetal heart shows prominent labeling in the ventricular muscle wall as well as very high levels of expression in the smooth muscle of the aorta and pulmonary vessels. Bar, 100 μm (in F).

Figure 8

β-Netrin RNA appears not to be expressed in the floorplate of midgestation mouse embryos. (A and B) nonradioactive in situ hybridizations of floor- and roofplate markers (netrin-1, and wnt-1) show good localization of transcriptions to these structures, respectively. (C and D) Two different probes for β-netrin, on the other hand, fail to localize to any region in embryonic day 11.5 spinal cord; in D we see some above background reactivity in the lateral margins of the floorplate.

Figure 9

β-Netrin is expressed in the adult rat olfactory bulb. (A) Heavy in situ reaction product is observed over the lateral olfactory tract, the output pathway from the bulb (arrowhead, Lat Olf Tract); in the periglomular cells (arrowhead, Glom L); and in mitral cell layer, the projection neurons of the bulb. Some reaction product is observed in the ventricles, ependyma and perhaps the blood vessel epithelium. (B and C) Protein localization by indirect immunofluorescence shows immunoreactivity in the lateral olfactory tract and the perineurum of the vomeronasal nerve (V-Nn). In addition, considerable immunoreactivity is observed in the basement membrane of the blood vessels.

Figure 10

β-Netrin produces neurite outgrowth from olfactory bulb explants. (A–C) Representative cultures treated as indicated. Treatment with rβNΔC or full-length rβN proteins increases the number of neurites produced by the explants. (D) Quantification of data obtained on neurite outgrowth; the total length of all neurites produced by explants is given here (Sum Length). Protein additions are noted below the bars; the line indicates that cultures were treated identically; the concentrations of protein additions are noted below the line. β-Netrin conventions are as above; 14 nM + AB indicates the simultaneous addition of 14 nM βN and pooled polyclonal antisera to βN. γ2SA denotes the short arm of the laminin γ2 chain; similar conventions are used for γ3 and β2 chains. * Statistically significant difference versus control; # statistically significant difference versus 14 nM βN or 9 nM γ3SA, respectively; P < 0.05.

Figure 11

Cartoon of laminin and netrin families. Three classes of netrin molecules have been described; netrins 1–3 are more related to laminin γ chains, whereas the netrin reported here, β-netrin, is more related to the laminin β chains. Two additional molecules, one of which is a putative transmembrane molecule and are homologous to the short arm of laminin chains, have been found (see text for details; for simplicity they are shown along side the α chain). Thus, there is a growing family of molecules which have homology to the short arms of laminin molecules.

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A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization - PubMed (original) (raw)