Biomedical subjects

Hans Merzendorfer

Publications and source records attributed to Hans Merzendorfer.

8 recordsLinked to original sources

PubMed2005

Insect chitin synthases: a review.

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP-N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes' catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.

Amino Acid Sequence↗

PubMed2005

Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development.

Chitin, the linear homopolymer of beta-1,4-linked N-acetylglucosamine, is produced by the enzyme chitin synthase (CHS). In general, this insoluble polysaccharide is found in two major extracellular structures in insects, the cuticle that overlays the epidermis and the peritrophic membrane (PM) that lines the midgut. Based on amino acid sequence similarities, insect CHSs are divided into two classes, A and B, and to date no more than two CHS genes have been identified in any single insect species. In species where both CHSs have been identified, one class A CHS and one class B CHS are always present. This finding suggests that these two genes may encode enzymes that synthesize chitin in different epithelial tissues. In our laboratory, we previously characterized transcripts for a class A CHS gene (MsCHS1) from the tobacco hornworm, Manduca sexta. We observed the expression of this gene in the larval epidermis, suggesting that the encoded enzyme functions to synthesize cuticular chitin. In this paper, we characterize a second chitin synthase gene (MsCHS2) belonging to class B and its cDNA from Manduca and show that it is expressed only in the midgut. This cDNA contains an open reading frame of 4575 nucleotides, which encodes a conceptual protein that is 1524 amino acids in length and is predicted to contain 16 transmembrane spans. Northern blot analysis of RNA isolated from anterior, medial, and posterior sections of the midgut from feeding larvae indicate that MsCHS2 is primarily expressed in the anterior midgut, with transcript levels tapering off in the medial and posterior midgut. Analysis of the MsCHS2 gene sequence indicates the absence of an alternate exon in contrast to the MsCHS1 gene, which yields two transcripts, MsCHS1a and MsCHS1b. RT-PCR analysis of the differential expression of these alternately spliced transcripts reveals that both splice variants are present in the epidermis. However, the ratio of the two alternately spliced transcripts varies during development, with MsCHS1a being generally more predominant. Southern blot analysis using a probe specific for CHS indicated that Manduca has only two CHS genes, akin to other insect species. Results from an analysis of expression of both genes in different tissues and developmental times indicate that the MsCHS1 enzyme is used for the synthesis of chitin in the cuticle and tracheae, whereas MsCHS2 is utilized exclusively for the synthesis of PM-associated chitin in the midgut.

Alternative Splicing↗

PubMed2004

The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments.

Previously, we have shown that the V-ATPase holoenzyme as well as the V1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V1 subunits B and C (Vitavska, O., Wieczorek, H., and Merzendorfer,H. (2003) J. Biol. Chem. 278, 18499-18505). Since the recombinant subunit C not only enhances actin binding of the V1 complex but also can bind separately to F-actin, we analyzed the interaction of recombinant subunit C with actin. We demonstrate that it binds not only to F-actin but also to monomeric G-actin. With dissociation constants of approximately 50 nm, the interaction exhibits a high affinity, and no difference could be observed between binding to ATP-G-actin or ADP-G-actin, respectively. Unlike other proteins such as members of the ADF/cofilin family, which also bind to G- as well as to F-actin, subunit C does not destabilize actin filaments. On the contrary, under conditions where the disassembly of F-actin into G-actin usually occurred, subunit C stabilized F-actin. In addition, it increased the initial rate of actin polymerization in a concentration-dependent manner and was shown to cross-link actin filaments to bundles of varying thickness. Apparently bundling is enabled by the existence of at least two actin-binding sites present in the N- and in the C-terminal halves of subunits C, respectively. Since subunit C has the possibility to dimerize or even to oligomerize, spacing between actin filaments could be variable in size.

Actin Cytoskeleton↗

PubMed2003

A novel role for subunit C in mediating binding of the H+-V-ATPase to the actin cytoskeleton.

Primary proton transport by V-ATPases is regulated via the reversible dissociation of the V(1)V(0) holoenzyme into its V(1) and V(0) subcomplexes. Laser scanning microscopy of different tissues from the tobacco hornworm revealed co-localization of the holoenzyme and F-actin close to the apical membranes of the epithelial cells. In midgut goblet cells, no co-localization was observed under conditions where the V(1) complex detaches from the apical membrane. Binding studies, however, demonstrated that both the V(1) complex and the holoenzyme interact with F-actin, the latter with an apparently higher affinity. To identify F-actin binding subunits, we performed overlay blots that revealed two V(1) subunits as binding partners, namely subunit B, resembling the situation in the osteoclast V-ATPase (Holliday, L. S., Lu, M., Lee, B. S., Nelson, R. D., Solivan, S., Zhang, L., and Gluck, S. L. (2000) J. Biol. Chem. 275, 32331-32337), but, in addition, subunit C, which gets released during reversible dissociation of the holoenzyme. Overlay blots and co-pelleting assays showed that the recombinant subunit C also binds to F-actin. When the V(1) complex was reconstituted with recombinant subunit C, enhanced binding to F-actin was observed. Thus, subunit C may function as an anchor protein regulating the linkage between V-ATPase and the actin-based cytoskeleton.

Actins↗

PubMed2003

The insect plasma membrane H+ V-ATPase: intra-, inter-, and supramolecular aspects.

The plasma membrane H+ V-ATPase from the midgut of larval Manduca sexta, commonly called the tobacco hornworm, is the sole energizer of epithelial ion transport in this tissue, being responsible for the alkalinization of the gut lumen up to a pH of more than 11 and for any active ion movement across the epithelium. This minireview deals with those topics of our recent research on this enzyme that may contribute novel aspects to the biochemistry and physiology of V-ATPases. Our research approaches include intramolecular aspects such as subunit topology and the inhibition by macrolide antibiotics, intermolecular aspects such as the hormonal regulation of V-ATPase biosynthesis and the interaction of the V-ATPase with the actin cytoskeleton, and supramolecular aspects such as the interactions of V-ATPase, K+/H+ antiporter, and ion channels, which all function as an ensemble in the transepithelial movement of potassium ions.

Animals↗

PubMed2003

Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases.

Chitin is one of the most important biopolymers in nature. It is mainly produced by fungi, arthropods and nematodes. In insects, it functions as scaffold material, supporting the cuticles of the epidermis and trachea as well as the peritrophic matrices lining the gut epithelium. Insect growth and morphogenesis are strictly dependent on the capability to remodel chitin-containing structures. For this purpose, insects repeatedly produce chitin synthases and chitinolytic enzymes in different tissues. Coordination of chitin synthesis and its degradation requires strict control of the participating enzymes during development. In this review, we will summarize recent advances in understanding chitin synthesis and its degradation in insects.

Animals↗

PubMed2002

Immunolocalization of chitin synthase in the tobacco hornworm.

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.

Amino Acid Sequence↗

PubMed2002

Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids.

V-ATPases are complex proteins consisting of a peripheral, ATP-hydrolysing V(1) complex and a membrane-bound H(+)-translocating V(o) complex. The plasma membrane V-ATPase from the tobacco hornworm (Manduca sexta) midgut is made up of eight different V(1) and four different V(o) subunits. During starvation and moulting, V-ATPase activity decreases as a result of the dissociation of the V(1) complex from the V(o) complex. To determine whether subunit biosynthesis is reduced during periods of enzyme inactivity, we measured the transcript levels and transcriptional activities of V-ATPase genes. Northern blots revealed the downregulation of almost all V-ATPase transcripts during starvation. During moulting, transcript levels of the three V-ATPase genes examined, mvB, mvG and mvd, also decreased, and this decrease was negatively correlated with the titre of 20-hydroxyecdysone (20-HE) and positively correlated with the titre of juvenile hormone (JH). To test the biological significance of these correlations, we injected both hormones into feeding larvae and measured transcript levels several hours later. A short-term increase and a long-term decrease in levels of mRNA were observed after 20-HE injection, whereas JH injection had no significant effect. Immunohistochemical studies of the midgut epithelium revealed that 20-HE injection led to changes in goblet cell morphology and in the subcellular distribution of the V(1) complex comparable with the situation during the moult and during starvation. Reporter gene assays in Sf21 cells using mvB, mvG and mvd promoters to initiate transcription of firefly luciferase led, after incubation of the cells with 20-HE, to results comparable with those obtained in the injection experiments. These findings suggest that putative ecdysone-responsive elements are present in all three promoters. Taken together, our results suggest that the expression of V-ATPase genes is controlled in a coordinated manner by ecdysteroids.

Animals↗