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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.

Koch M, Murrell JR, Hunter DD, Olson PF, Jin W, Keene DR, Brunken WJ, Burgeson RE - J. Cell Biol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

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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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). Mr, 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.
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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). Mr, 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.

Mentions: The calculated mass of rβ-N + His is 68 kD; it migrates with an electrophoretic mobility predicting a final mass of 70 kD (Fig. 4 A). rβ-NΔC + His has an electrophoretic mobility consistent with a mass of 56 kD (Fig. 4 A), which corresponds well with a predicted mass of 53 kD. Removal of the His tag by thrombin cleavage reduces the apparent molecular mass slightly, as expected. rβ-N + His and rβ-NΔC + His, as well as recombinant laminin β2 short arm containing a His tag are all recognized by an anti-His antibody, but there is no residual activity against rβ-N after removal of the His tag (Fig. 4 B).


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

Koch M, Murrell JR, Hunter DD, Olson PF, Jin W, Keene DR, Brunken WJ, Burgeson RE - J. Cell Biol. (2000)

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). Mr, 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192657&req=5

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). Mr, 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.
Mentions: The calculated mass of rβ-N + His is 68 kD; it migrates with an electrophoretic mobility predicting a final mass of 70 kD (Fig. 4 A). rβ-NΔC + His has an electrophoretic mobility consistent with a mass of 56 kD (Fig. 4 A), which corresponds well with a predicted mass of 53 kD. Removal of the His tag by thrombin cleavage reduces the apparent molecular mass slightly, as expected. rβ-N + His and rβ-NΔC + His, as well as recombinant laminin β2 short arm containing a His tag are all recognized by an anti-His antibody, but there is no residual activity against rβ-N after removal of the His tag (Fig. 4 B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

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.

Show MeSH
Related in: MedlinePlus