Immunolocalization of chitin synthase in the tobacco hornworm

Abstract

To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

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

“…Polyclonal antibodies to the recombinant catalytic domain of the Manduca chitin synthase were generated previously (Zimoch and Merzendorfer, 2002). To generate anti-CTLP1 antibodies, the peptide IVGGTQAPSGSHPH (amino acid positions 41-54) was synthesized, coupled to Keyhole limpet haemocyanin and used for the immunization of rabbits (Charles River, Sulzfeld, Germany).…”

“…Blot membranes were stained with 0.02% (v/v) Ponceau S (Sigma, Taufkirchen, Germany). Immunoblots were performed as described previously (Zimoch and Merzendorfer, 2002). The immunoreactions were carried out with polyclonal anti-CHS antibodies (1:1000 dilution), anti-CTLP1 antibodies (1:1000 dilution) or monoclonal antibodies to the V-ATPase subunit A [1:10 dilution; also named 221-9 (Klein et al, 1991)].…”

“…The secondary antibodies were anti-rabbit or anti-mouse antibodies (1:10·000 and 1:30·000 dilution, respectively; whole molecules conjugated with alkaline phosphatase, Sigma). Cryosectioning of tissues and immunocytochemistry were carried out as described previously (Zimoch and Merzendorfer, 2002). Immunoprecipitation was performed with the protein G Immunoprecipitation kit (Sigma) following the instructions of the manufacturer.…”

“…Protein expression was performed in E. coli Rosetta (DE3) pLysS cells as described previously (Merzendorfer et al, 2000). Synthesis of RNA probes and in situ hybridization were performed according to previous protocols (Zimoch and Merzendorfer, 2002), with the exception that the detection of RNA probes was carried out with anti-Dig-gold antibodies (Roche) and silver enhancement (IntenSE TM silver enhancement kit, GE Amersham). RNA probes were complementary to the cDNA nucleotide positions 1-178 of CTPL1 (GenBank accession no.…”

“…Immunocytochemistry has revealed that the chitin synthase from feeding larvae is localized at the very apical tips of the brush border microvilli formed by midgut columnar cells (Zimoch and Merzendorfer, 2002). As well as transcriptional regulation of chitin synthase expression, post-translational mechanisms also appear to be involved in regulating the enzyme.…”