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Teneurin-1 is expressed in interconnected regions of the developing brain and is processed in vivo.

Kenzelmann D, Chiquet-Ehrismann R, Leachman NT, Tucker RP - BMC Dev. Biol. (2008)

Bottom Line: Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information.Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstr. 66, 4057 Basel, Switzerland. daniela.kenzelmann@fmi.ch

ABSTRACT

Background: Teneurins are a unique family of transmembrane proteins conserved from C. elegans and D. melanogaster to mammals. In vertebrates there are four paralogs (teneurin-1 to -4), all of which are expressed prominently in the developing central nervous system.

Results: Analysis of teneurin-1 expression in the developing chick brain by in situ hybridization and immunohistochemistry defined a unique, distinct expression pattern in interconnected regions of the brain. Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information. Based on these expression patterns, we suspect a role for teneurins in neuronal connectivity. In contrast to the cell-surface staining of the antibody against the extracellular domain, an antibody recognizing the intracellular domain revealed nuclear staining in subpopulations of neurons and in undifferentiated mesenchyme. Western blot analysis of brain lysates showed the presence of N-terminal fragments of teneurin-1 containing the intracellular domain indicating that proteolytic processing occurs. Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.

Conclusion: Teneurin-1 and -2 are expressed by distinct interconnected populations of neurons in the developing central nervous system. Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

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Teneurin-1 appears to be processed in the developing chicken brain. Western blot analysis of embryonic chicken brain lysates. Detection of endogenous teneurin-1 using anti-ICD (A) and anti-ECD antibodies (B). The numbers to the left indicate the positions of molecular weight standards. The anti-ICD detects a prominent N-terminal fragment at 65 kDa (arrowhead), which is much smaller than expected for the full-length protein (arrow), suggesting proteolytic processing. In contrast, using the anti-ECD antibody results in detection of the full-length protein (arrow) and a large C-terminal fragment (arrowhead). Asterisks label additional processing products (see text for details). Postulated cleavage sites and calculated molecular weights of processing products (C). Postulated cleavage sites: 1 = ectodomain shedding, 2 = intramembrane cleavage releasing the ICD. The sizes indicated are predicted from the amino acid sequence; known apparent molecular weights are shown in parentheses (see text for details). The calculated molecular weights are approximate, as the exact cleavage sites are not known.
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Figure 4: Teneurin-1 appears to be processed in the developing chicken brain. Western blot analysis of embryonic chicken brain lysates. Detection of endogenous teneurin-1 using anti-ICD (A) and anti-ECD antibodies (B). The numbers to the left indicate the positions of molecular weight standards. The anti-ICD detects a prominent N-terminal fragment at 65 kDa (arrowhead), which is much smaller than expected for the full-length protein (arrow), suggesting proteolytic processing. In contrast, using the anti-ECD antibody results in detection of the full-length protein (arrow) and a large C-terminal fragment (arrowhead). Asterisks label additional processing products (see text for details). Postulated cleavage sites and calculated molecular weights of processing products (C). Postulated cleavage sites: 1 = ectodomain shedding, 2 = intramembrane cleavage releasing the ICD. The sizes indicated are predicted from the amino acid sequence; known apparent molecular weights are shown in parentheses (see text for details). The calculated molecular weights are approximate, as the exact cleavage sites are not known.

Mentions: Western blot analysis using the ICD antibody showed a prominent band at 65 kDa (Fig. 4A, arrowhead), which is much smaller than expected for the full length protein (> 300 kDa). The specificity was confirmed by competition with the ICD peptide which completely abrogates detection of this band (not shown). However, 65 kDa is larger than we would expect for the teneurin-1 ICD, because an N-terminal fragment containing the amino acids up to the transmembrane domain (ICD construct) migrates at 45 kDa [21]. Therefore, this fragment may still contain the transmembrane domain. Such an N-terminal fragment would be expected to occur after ectodomain shedding (Fig. 4C, cleavage 1). The full-length protein was barely detectable (Fig. 4A, arrow), indicating that ectodomain shedding may take place constitutively. The faint bands observed below the 65 kDa band (Fig. 4A, asterisks) at approximately 50 and 35 kDa might be the released ICD generated after the second cleavage (Fig. 4C, cleavage 2), which occurs only in a specific subset of cells. These data suggest that proteolytic processing of the full-length protein takes place in vivo, resulting in N-terminal fragments. When the same extracts were analyzed using an antibody detecting the ECD (Fig. 4B), a faint band > 250 kDa (arrow) and a stronger band around 250 kDa (arrowhead) are observed. Since the predicted molecular weight of full-length teneurins is more than 300 kDa, the upper band may represent the full-length protein, and the lower band could correspond to the ECD after shedding, which has a predicted molecular weight of 245 kDa (Fig. 4C). The smear above these bands can be explained by glycosylation of the extracellular part of the protein [7]. The band at 150 kDa (Fig. 4B, asterisk) could be a further cleaved extracellular fragment of teneurin-1. In Figure 4C the postulated cleavage sites and the resulting fragments are summarized. Note that the molecular weights are either calculated based on the amino sequences or based on observations (shown in parentheses). The ICD has a higher apparent molecular weight than predicted, therefore we expect the same to be the case for the 65 kDa N-terminal fragment, for which the predicted molecular weight would only be 56 kDa (Fig. 4C).


Teneurin-1 is expressed in interconnected regions of the developing brain and is processed in vivo.

Kenzelmann D, Chiquet-Ehrismann R, Leachman NT, Tucker RP - BMC Dev. Biol. (2008)

Teneurin-1 appears to be processed in the developing chicken brain. Western blot analysis of embryonic chicken brain lysates. Detection of endogenous teneurin-1 using anti-ICD (A) and anti-ECD antibodies (B). The numbers to the left indicate the positions of molecular weight standards. The anti-ICD detects a prominent N-terminal fragment at 65 kDa (arrowhead), which is much smaller than expected for the full-length protein (arrow), suggesting proteolytic processing. In contrast, using the anti-ECD antibody results in detection of the full-length protein (arrow) and a large C-terminal fragment (arrowhead). Asterisks label additional processing products (see text for details). Postulated cleavage sites and calculated molecular weights of processing products (C). Postulated cleavage sites: 1 = ectodomain shedding, 2 = intramembrane cleavage releasing the ICD. The sizes indicated are predicted from the amino acid sequence; known apparent molecular weights are shown in parentheses (see text for details). The calculated molecular weights are approximate, as the exact cleavage sites are not known.
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Related In: Results  -  Collection

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Figure 4: Teneurin-1 appears to be processed in the developing chicken brain. Western blot analysis of embryonic chicken brain lysates. Detection of endogenous teneurin-1 using anti-ICD (A) and anti-ECD antibodies (B). The numbers to the left indicate the positions of molecular weight standards. The anti-ICD detects a prominent N-terminal fragment at 65 kDa (arrowhead), which is much smaller than expected for the full-length protein (arrow), suggesting proteolytic processing. In contrast, using the anti-ECD antibody results in detection of the full-length protein (arrow) and a large C-terminal fragment (arrowhead). Asterisks label additional processing products (see text for details). Postulated cleavage sites and calculated molecular weights of processing products (C). Postulated cleavage sites: 1 = ectodomain shedding, 2 = intramembrane cleavage releasing the ICD. The sizes indicated are predicted from the amino acid sequence; known apparent molecular weights are shown in parentheses (see text for details). The calculated molecular weights are approximate, as the exact cleavage sites are not known.
Mentions: Western blot analysis using the ICD antibody showed a prominent band at 65 kDa (Fig. 4A, arrowhead), which is much smaller than expected for the full length protein (> 300 kDa). The specificity was confirmed by competition with the ICD peptide which completely abrogates detection of this band (not shown). However, 65 kDa is larger than we would expect for the teneurin-1 ICD, because an N-terminal fragment containing the amino acids up to the transmembrane domain (ICD construct) migrates at 45 kDa [21]. Therefore, this fragment may still contain the transmembrane domain. Such an N-terminal fragment would be expected to occur after ectodomain shedding (Fig. 4C, cleavage 1). The full-length protein was barely detectable (Fig. 4A, arrow), indicating that ectodomain shedding may take place constitutively. The faint bands observed below the 65 kDa band (Fig. 4A, asterisks) at approximately 50 and 35 kDa might be the released ICD generated after the second cleavage (Fig. 4C, cleavage 2), which occurs only in a specific subset of cells. These data suggest that proteolytic processing of the full-length protein takes place in vivo, resulting in N-terminal fragments. When the same extracts were analyzed using an antibody detecting the ECD (Fig. 4B), a faint band > 250 kDa (arrow) and a stronger band around 250 kDa (arrowhead) are observed. Since the predicted molecular weight of full-length teneurins is more than 300 kDa, the upper band may represent the full-length protein, and the lower band could correspond to the ECD after shedding, which has a predicted molecular weight of 245 kDa (Fig. 4C). The smear above these bands can be explained by glycosylation of the extracellular part of the protein [7]. The band at 150 kDa (Fig. 4B, asterisk) could be a further cleaved extracellular fragment of teneurin-1. In Figure 4C the postulated cleavage sites and the resulting fragments are summarized. Note that the molecular weights are either calculated based on the amino sequences or based on observations (shown in parentheses). The ICD has a higher apparent molecular weight than predicted, therefore we expect the same to be the case for the 65 kDa N-terminal fragment, for which the predicted molecular weight would only be 56 kDa (Fig. 4C).

Bottom Line: Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information.Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstr. 66, 4057 Basel, Switzerland. daniela.kenzelmann@fmi.ch

ABSTRACT

Background: Teneurins are a unique family of transmembrane proteins conserved from C. elegans and D. melanogaster to mammals. In vertebrates there are four paralogs (teneurin-1 to -4), all of which are expressed prominently in the developing central nervous system.

Results: Analysis of teneurin-1 expression in the developing chick brain by in situ hybridization and immunohistochemistry defined a unique, distinct expression pattern in interconnected regions of the brain. Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information. Based on these expression patterns, we suspect a role for teneurins in neuronal connectivity. In contrast to the cell-surface staining of the antibody against the extracellular domain, an antibody recognizing the intracellular domain revealed nuclear staining in subpopulations of neurons and in undifferentiated mesenchyme. Western blot analysis of brain lysates showed the presence of N-terminal fragments of teneurin-1 containing the intracellular domain indicating that proteolytic processing occurs. Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.

Conclusion: Teneurin-1 and -2 are expressed by distinct interconnected populations of neurons in the developing central nervous system. Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

Show MeSH