spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online October 5, 2007
Journal of Experimental Biology 210, 3636-3643 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008334
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Broehan, G.
Right arrow Articles by Merzendorfer, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Broehan, G.
Right arrow Articles by Merzendorfer, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

Gunnar Broehan, Lars Zimoch, Anton Wessels, Beyhan Ertas and Hans Merzendorfer*

Department of Biology/Chemistry, University of Osnabrück, D-49069 Osnabrück, Germany


Figure 1
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 1. Domain architecture of the midgut chitin synthase CHS2. The two black horizontal lines represent the apical brush border membrane of midgut columnar cells. Horizontal bars represent intra- or extracellular domains and vertical bars transmembrane helices. (A) Aminoterminal transmembrane region; (B) cytoplasmic catalytic domain; (C) carboxyterminal transmembrane region; the numbers refer to single domains within each region. Those domains that were tested as baits in the yeast two-hybrid system are coloured dark grey. UDP, uridine 5'-diphosphate; GlcNAc, N-acetyl-D-glucosamine.

 

Figure 2
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 2. Primary structure of CTPL1. cDNA sequence and deduced amino acid sequence (standard: zymogen; bold: mature protease) of the M. sexta CTLP1. Italic characters indicate the 5' and 3' untranslated regions of the cDNA and the putative signal peptide for the secretory pathway of the protein. Arrows mark the predicted cleavage sites of the signal peptidase and trypsin.

 

Figure 3
View larger version (93K):
[in this window]
[in a new window]

  		Fig. 3. ClustalW alignment of insect chymotrypsin-like proteases. Amino acids that are conserved, highly conserved or identical in all sequences are highlighted in light grey, grey or black, respectively. Open triangle, conserved trypsin cleavage site; filled triangles, conserved residues of the S1 specificity pocket; arrows, residues of the catalytic triad of serine proteases; asterisks, conserved cysteines; bar, putative N-glycosylation site for CTLP1. Ms, Manduca sexta; Ag, Anopheles gambiae; Dm, Drosophila melanogaster; CT, chymotrypsin.

 

Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 4. Expression of CTLP1 and CHS2 in the midgut of M. sexta. Poly(A) RNA from the anterior (a), median (m) and posterior (p) midgut was reverse transcribed and used as a template for PCR (25 cycles) to amplify the cDNAs encoding CTLP1 and CHS2. Control reactions showed the absence of contaminating RNA and DNA (not shown).

 

Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 5. Localization of CTLP1 and CHS2 mRNAs in the anterior midgut of M. sexta. In situ hybridization was performed under high stringency conditions using 20 µm cryosections of the anterior midgut and antisense RNA probes to detect the sense RNAs for CTLP1 and CHS2 (left). As a negative control, sense RNA probes were use to detect the corresponding antisense RNAs (right).

 

Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 6. Immunoblots demonstrating that CTLP1 is secreted and proteolytically processed. Proteins from CTLP1-expressing bacterial cells (1), the gut contents (2) and the anterior midgut (3) were separated by SDS-PAGE, blotted onto nitrocellulose and stained with monospecific antibodies to CTLP1. Standard proteins with molecular masses indicated in kDa are also shown (S).

 

Figure 7
View larger version (65K):
[in this window]
[in a new window]

  		Fig. 7. Immunolocalization of CTLP1 and CHS2 in the midgut of M. sexta. Midgut cryosections of 10 µm thickness from fifth instar larvae were stained with either anti-CTLP1 antibodies (A) or anti-CHS antibodies (B) directed to the recombinant catalytic domain of chitin synthase (Zimoch and Merzendorfer, 2002Go). Arrows mark the apical brush border of the midgut epithelium, the asterisk marks immunoreactive material in the gut lumen. The insets show corresponding negative controls performed in the absence of primary antibodies. a, apical; b, basal.

 

Figure 8
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 8. Co-immunoprecipitation of CTLP1 and CHS2. For immunoprecipitation, cell lysates of the anterior midgut were incubated with the indicated precipitating antibodies (P) and then bound to protein-G–agarose. Unbound proteins were washed away, then bound proteins were eluted, separated by SDS-PAGE and analysed by immunoblotting using detecting antibodies (D) to CTLP1, CHS2 and V-ATPase subunit A (V1A). Lanes 1–4, co-immunoprecipitation of CHS2 using anti-CTLP1 antibodies (lanes 1,2) and of CTLP1 using anti-CHS antibodies (lanes 3,4); lanes 5–8, control reactions in the absence of precipitating (lanes 5,6) or detecting antibodies (lanes 7,8); lanes 9,10, control reactions for non-specific precipitation of V-ATPase. As a positive control, midgut cell lysates were used (lane 11).

 

Figure 9
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 9. Stimulation of chitin synthesis in midgut extracts by serine proteases. Relative chitin synthase activity was measured by the incorporation of N-acetyl-D-[U-14C]glucosamine into alkali-insoluble material obtained from crude extracts of the anterior midgut of fifth instar larvae. (1) Activities of untreated extracts (0.4 nmol mg–1 h–1) were set to 100%; (2–4) relative activities in the presence of trypsin at a concentration of 10 µg µl–1 (2), chymotrypsin at a concentration of 10 µg µl–1 (3), and 5 mmol l–1 Pefabloc SC serine protease inhibitor (4). Mean values (±s.e.m.) from three independent experiments are given as a percentage of the control.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007