A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae

The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca2+ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca2+, a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a two-step limited proteolysis in the presence of Ca2+.     

A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.

To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.     

Physical and catalytic properties of alpha-amylase from Tenebrio molitor L. larvae.

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an alpha-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect alpha-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch.     

The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the beta-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the (3)J(alphabeta) (1)H-(1)H scalar coupling constants, the chi(1) angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.     

Characteristic properties of proteins from pre-ecdysial cuticle of larvae and pupae of the mealworm Tenebrio molitor

Proteins extracted from the cuticle of pharate larvae and pupae of the mealworm Tenebrio molitor are more soluble at low temperatures than at higher temperatures, a behaviour characteristic of hydrophobic proteins. When the temperature of an unfractionated cuticular extract is raised from 4 to 25 degrees C the solution becomes turbid, droplets of a heavy, protein-rich phase are formed, which gradually settles, leaving an upper protein-poor phase, indicating that the aggregation process is a coacervation. The aggregation of the dissolved cuticular proteins is influenced by changes in temperature, pH, and ionic strength. The process has been studied by measuring development of turbidity in unfractionated cuticular extracts and in solutions of three purified proteins from Tenebrio pharate larvae and pupae (TmLPCP-A1a, TmLPCP-E1a, and TmLPCP-G1a), while temperature, pH or ionic strength of the solutions were varied. Protein aggregation was also studied by determination of changes in fluorescence intensity, when the hydrophobicity probe, 8-anilinonaphthalenesulfonic acid (ANS) was added to solutions of the cuticular proteins. Only when the protein solutions had developed a measurable turbidity was an increase in ANS-fluorescence observed, indicating formation of tightly packed clusters of hydrophobic amino acid residues during aggregation. The temperature range for aggregation depends upon protein concentration: the higher the concentration the lower and more narrow is the temperature range within which aggregation occurs. The tendency for the individual cuticular proteins to aggregate is most pronounced near their isoelectric points, and most of the cuticular proteins have alkaline isoelectric points. The influence of salts on the tendency of the proteins to aggregate varies among the proteins and depends upon how close they are to their isoelectric point. A solution containing both protein TmLPCP-A1a and TmLPCP-E1a becomes more turbid and develops a more intense ANS-fluorescence when warmed from 10 to 30 degrees C than corresponding to the sum of measurements performed on separate solutions of the two proteins, indicating that the two proteins interact during aggregation. The Tenebrio larval/pupal cuticular proteins are characterized by an abundance of hydrophobic amino acid residues, and especially their contents of alanine and proline are high. The behaviour of the cuticular proteins in solution resembles that of another hydrophobic protein, tropoelastin, and it seems reasonable to suggest that similar interactions govern the folding and aggregation of the peptide chains in the two types of proteins. The proline and alanine rich chain segments in the pharate cuticular proteins are suggested to form a series of beta-turns and to fold into a relatively open structure at low temperatures, giving water access to the hydrophobic residues and making the proteins water soluble. At increased temperatures the structure of the ordered water layer surrounding the hydrophobic groups breaks down, and the peptide chains tend to collapse into a more closed structure and to interact more tightly with hydrophobic regions in neighbouring molecules. In dilute solutions in the test tube this results in aggregation and precipitation of the proteins; in intact, pharate cuticle at ambient temperatures the proteins will preferably be in an aggregated, easily dissociated state. Accordingly, small changes in intercuticular pH and ionic strength can produce pronounced changes in the mechanical properties of unsclerotized solid cuticle by interference with protein interactions, in agreement with reports that some cuticles undergo plasticization during and/or immediately after ecdysis.     

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.     

Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type

Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.     

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine.

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and beta-1, 3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and beta-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.     

Characterization of a calcium-activated protease that hydrolyzes a microtubule-associated protein

Rat brain extract contains a calcium-activated protease that hydrolyzes the high-molecular-weight microtubule-associated protein MAP₂. We have purified this enzyme greater than 30-fold by pH precipitation, DEAE, and Bio-Gel P-300 chromatography. The partially purified enzyme is half-maximally activated by 2 μm calcium when assayed using 2× cycled microtubules as substrate. The enzyme is active over a pH range of 6.5 to 7.5 and is heat labile and temperature dependent. The calcium-dependent regulator, calmodulin, cannot replace the protease in promoting MAP₂ hydrolysis. The partial purification of a calcium-activated protease from brain tissue explains the sensitivity of in vitro tubulin polymerization to micromolar concentrations of calcium.     

Lipid nutritional indices,regioisomeric distribution,and thermal properties of Tenebrio molitor and Hermetia illucens larvae fat

The fatty acid (FA) profile, nutritional index, and thermal properties of lipids from Tenebrio molitor and Hermetia illucens larvae were studied. T. molitor and H. illucens larvae had high lipid contents (respectively 28.8% and 42.6%), saturated (25.0% and 55.8%), monounsaturated (MUFA) (39.2% and 28.3%), and polyunsaturated (PUFA) fatty acids (35.8% and 15.9%). Both larvae fats contained beneficial ω-3, ω-6, and ω-9 FA. For T. molitor and H. illucens, the lipid nutritional indices were atherogenicity indices 0.68 and 2.75, thrombogenicity indices 0.58 and 0.74, and health-promoting indices 3.51 and 0.80 hypocholesterolemic/hypercholesterolemic acid ratio 1.38 and 0.23, suggesting the nutritional superiority of T. molitor larvae fat. Regioisomeric distribution analysis showed that PUFA in H. illucens larvae fat are concentrated on the sn-1,3 positions, whereas those in T. molitor larvae fat are distributed in all three positions. The thermal stability and crystallisation profiles differed for both larvae fats and demonstrated their potential use in thermally processed foods.     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.     

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.     

Characterization of a novel protein that specifically binds to DNA modified by N-acetoxy-acetylaminofluorene and cis-diamminedichloroplatinum

Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as a substrate and electrophoretic mobility shift and Southwestern blot assays. One major damage-recognizing protein was detected, whose amount was estimated as at least 10(5) copies per cell. Levels of this protein were similar in extracts from brain, kidney and liver, but much lower in extracts from testis. The affinity of the detected protein for DNA damaged by AAAF was about 70-fold higher than for undamaged DNA. DNA damaged by cis-diamminedichloroplatinum (cis-DDP), benzo(a)pyrene diolepoxide (BPDE) or UV-radiation also bound this protein with an increased affinity, the former more strongly and the latter two more weakly as compared to AAAF-damaged DNA. The detected AAAF/DDP-damaged-DNA-binding (AAAF/DDP-DDB) protein had a molecular mass of about 25 kDa and was distinct from histone H1 or HMGB proteins, which are known to have a high affinity for cis-DDP-damaged DNA. The level of this damage-recognizing protein was not affected in rats treated with the carcinogen 2-acetylaminofluorene. The activity of an AAAF/DDP-DDB protein could also be detected in extracts from mouse liver cells but not from the Hep2G human hepatocellular carcinoma.     

Developmental expression and hormonal regulation of a desiccation stress protein in Tenebrio molitor

In a previous study it has been reported that dsp28 is induced during desiccation in Tenebrio larvae. During that study it was observed that in non-stressed larvae the concentration of dsp28 in hemolymph drops dramatically just prior to pupation. These results suggested that control of dsp28 synthesis is subject to environmental as well as hormonal cues. This study identifies a site of synthesis as fat body, as dsp28 was secreted into the medium during in vitro incubation of larval fat bodies. Using immunoelectrophoresis to determine protein concentration a developmental profile showing changes in levels of dsp28 in hemolymph during larval, pupal and adult stages of Tenebrio molitor was established. The concentration of dsp28 in larval hemolymph dropped from 4 to 5 mg/ml to 1.5 mg/ml just prior to pupation. This lower level was maintained until adults emerged when the concentration of dsp28 rose to prepupal levels again. Hormonal regulation is suggested since application of methoprene to newly-emerged pupae resulted in an increased incorporation of radiolabeled cysteine into dsp28.     

Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae.

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the biochemical process of encapsulation is still obscure. To isolate and characterize the early-stage encapsulation-relating protein (ERP), we used the coleopteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted pieces of surgical suture were recovered 10 min after injection or insertion, and were analyzed on SDS/PAGE under reducing conditions. Four different proteins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymph. Among them, we purified 56-kDa and 48-kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity-purified antibodies of the 56-kDa and 48-kDa ERPs cross-reacted with the 48-kDa and 56-kDa ERPs, respectively. Analysis of the cDNA of 56-kDa ERP consisted of 579 amino acid residues and showed a novel glutamine-rich protein. Positive clones of the 48-kDa ERP showed the same DNA sequence as 56-kDa ERP. Interestingly, the chemically determined N-terminal amino acid sequence and the three partial amino acid sequences of the 48-kDa protein were found in the 56-kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101-Gly102 of the 56-kDa ERP by a limited proteolysis. Western blotting analysis showed that these ERPs were detected exclusively on membrane fractions of hemocytes. Also, when the early-stage encapsulated beads were coated with both the 56-kDa and 48-kDa ERP antibodies and re-injected into larvae, no further encapsulation reaction was observed. However, when the early-stage encapsulated beads were incubated with 56-kDa ERP antibody, 48-kDa ERP antibody or nonimmunized rabbit IgG and re-injected into larvae, further encapsulation did occur.     

Secretion of beta-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae

There are four β-glycosidases (βgly1, βgly2, βgly3, and βgly4) in Tenebrio molitor midgut larvae. βgly1 and βgly2 have identical kinetic properties, and differ in a few amino acid residues. Purified βgly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only βgly1 and βgly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that βgly1+βgly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only βgly1+βgly2, revealed that most of those β-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry βgly1 and βgly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.     

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.     

Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia

We report the isolation and characterization of a cDNA encoding the novel mammalian serine protease Omi. Omi protein consists of 458 amino acids and has homology to bacterial HtrA endoprotease, which acts as a chaperone at low temperatures and as a proteolytic enzyme that removes denatured or damaged substrates at elevated temperatures. The carboxyl terminus of Omi has extensive homology to a mammalian protein called L56 (human HtrA), but unlike L56, which is secreted, Omi is localized in the endoplasmic reticulum. Omi has several novel putative protein-protein interaction motifs, as well as a PDZ domain and a Src homology 3-binding domain. Omi mRNA is expressed ubiquitously, and the gene is localized on human chromosome 2p12. Omi interacts with Mxi2, an alternatively spliced form of the p38 stress-activated kinase. Omi protein, when made in a heterologous system, shows proteolytic activity against a nonspecific substrate beta-casein. The proteolytic activity of Omi is markedly up-regulated in the mouse kidney following ischemia/reperfusion.