Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.     

Isolation and some properties of two deoxyribonucleases from a snail, Achatina fulica.

1. Two main DNases were found in the dried liver extract of a snail, Achatina fulica. They were purified by the phosphocellulose batch method and by phosphocellulose column chromatography. The enzyme eluted earlier from the phosphocellulose column was designated as Achatina DNase-1 and the other as Achatina DNase-2. DNase-1 was purified further by QAE-Sephadex A-25 column chromatography (twice) just before use because of the instability of the purified enzyme. By these procedures, DNase-1 and 2 were purified 200- and 130-fold, respectively. 2. Divalent or monovalent cations had no marked effect on either enzyme. The showed pH optima of 4.8 (DNase-1) and 5.2 (DNase-2). Ionic strength was found to be critical for the maximal activity. The isoelectric points of DNase-1 and 2 were both 6.9. On heating at 70--75 degrees C for 5 min, each enzymic activity fell to half of the initial value. 3. The enzyme preparations degraded native DNA 1.5--2.5 times faster than heat-denatured DNA. They both degraded heat-denatured DNA endonucleolytically, to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphoryl and 5'-hydroxy termini of the resulting oligonucleotides were analyzed. DNase-1 possessed marked specificity for dThd at 3'-termini and dAdo at 5'-termini in the early stages of degradation, but only for dAdo at 5'-termini in the later stages. DNase-2 showed some preference for purine nucleotides at both 3'- and 5'-termini in the later stages of degradation.     

Further characterization of the cold agglutinin from the snail Achatina fulica.

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.     

Studies on chemical modification of cold agglutinin from the snail Achatina fulica.

The cold agglutinin isolated from the albumin gland of the snail Achatina fulica was modified with various chemical reagents in order to detect the amino acids and/or carbohydrate residues present in its carbohydrate-binding sites. Treatment with reagents considered specific for modification of lysine, arginine and tryptophan residues of the cold agglutinin did not affect the carbohydrate-binding activity of the agglutinin. Modification of tyrosine residues showed some change. However, modification with carbodiimide followed by alpha-aminobutyric acid methyl ester causes almost complete loss of its binding activity, indicating the involvement of aspartic acid and glutamic acid in its carbohydrate-binding activity. The carbohydrate residues of the cold agglutinin were removed by beta-elimination reaction, indicating that the sugars are O-glycosidically linked to protein part of the molecule. Removal of galactose residues from the cold agglutinin by the action of beta-galactosidase indicated that the galactose molecules are beta-linked. These carbohydrate-modified glycoproteins showed a marked change in agglutination property, i.e. they agglutinated rabbit erythrocytes at both 10 degrees C and 25 degrees C, indicating that the galactose residues of the glycoprotein play an important role in the cold-agglutination property of the glycoprotein. The c.d. data showed the presence of an almost identical type of random-coil conformation in the native cold agglutinin at 10 degrees C and in the carbohydrate-modified glycoprotein at 10 degrees C and 25 degrees C. This particular random-coil conformation is essential for carbohydrate-binding property of the agglutinin.     

Gut bacterial communities in the giant land snail Achatina fulica and their modification by sugarcane-based diet

The invasive land snail Achatina fulica is one of the most damaging agricultural pests worldwide representing a potentially serious threat to natural ecosystems and human health. This species is known to carry parasites and harbors a dense and metabolically active microbial community; however, little is known about its diversity and composition. Here, we assessed for the first time the complexity of bacterial communities occurring in the digestive tracts of field-collected snails (FC) by using culture-independent molecular analysis. Crop and intestinal bacteria in FC were then compared to those from groups of snails that were reared in the laboratory (RL) on a sugarcane-based diet. Most of the sequences recovered were novel and related to those reported for herbivorous gut. Changes in the relative abundance of Bacteroidetes and Firmicutes were observed when the snails were fed a high-sugar diet, suggesting that the snail gut microbiota can influence the energy balance equation. Furthermore, this study represents a first step in gaining a better understanding of land snail gut microbiota and shows that this is a complex holobiont system containing diverse, abundant and active microbial communities.     

Effects of procaine on a central neuron of the snail, Achatina fulica Ferussac

Effects of procaine on a central neuron (RP1) of the giant African snail (Achatina fulica Ferussac) were studied pharmacologically. The RP1 neuron showed spontaneous firing of action potential. Extra-cellular application of procaine (10 mM) reversibly elicited bursts of potential. The bursts of potential elicited by procaine were not blocked after administration of (1) prazosin, propranolol, atropine, d-tubocurarine, (2) calcium-free solution, (3) ryanodine (4) pretreatment with KT-5720 or chelerythrine. The bursts of potential elicited by procaine were blocked by adding U73122 (10 microM) and the bursts of potential were decreased if physiological sodium ion was replaced with lithium ion or incubated with either neomycin (3.5 mM) or high magnesium solution (30 mM). Preatment with U73122 (10 microM) blocked the initiation of bursts of potential. Ruthenium red (100 microM) or caffeine (10 mM) facilitated the procaine-elicited bursts of potential. It is concluded that procaine reversibly elicits bursts of potential in the central snail neuron. This effect was not directly related to (1) the extra-cellular calcium ion fluxes, (2) the ryanodine sensitive calcium channels in the neuron, or (3) the PKC or PKA related messenger systems. The procaine-elicited bursts of potential were associated with the phospholipase activity and the calcium mobilization in the neuron.     

Fulicin regulates the female reproductive organs of the snail, Achatina fulica

Fulicin is a D-amino acid-containing neuropeptide that has been thought to control male copulatory behavior in the land snail, Achatina fulica. In the present study, we demonstrated that the vagina and the oviduct of Achatina were densely innervated by fulicin-like immunoreactive neuronal fibers. We confirmed that fulicin was actually present in the vagina by mass spectrometry. Furthermore, fulicin showed a profound excitatory effect on contractions of the vagina and the oviduct. These results suggest that fulicin controls female egg-laying behavior as an excitatory neuropeptide regulating the female reproductive organs of the snail.     

Induction of locomotor behavior in the giant African snail,Achatina fulica

• 1.1. The locomotor-inducting factor of the giant African snail, Achatina fulica, was examined.
• 2.2. Snails showed nocturnal circadian behavior in relative humidity at least over 50%. Although the rhythmicity was independent of light and darkness, it was disturbed easily by hydration, and hydrated snails continued to locomote throughout the day. For induction of locomotor behavior, relative humidity over 50% was the fundamental factor and water is shown to be the limiting factor for the endogeneous circadian oscillator.
• 3.3. The integument of snails showed a higher water permeability. Through the integument, hemolymph osmolality changed easily according to hydration and dehydration from about 120 to 400 mOsm/kg H₂O. Circadian behavior was induced in snails in which hemolymph osmolality ranged from about 130 to 230 mOsm/kg H₂O.
• 4.4. By hydration, hemolymph osmolality in quiescent and estivated snails which have higher osmolality decreased gradually and then they began to locomote according to the degree of dilution, and vice versa. The induction of behavior in these snails was controlled by low hemolymph osmolality.
• 5.5. Together with the endogeneous rhythmicity, water environment was shown to be the key factor for the induction of locomotor behavior.
• 6.6. Based on these results, the mechanisms of the induction of locomotor behavior in terrestrial pulmonates are proposed.

     

An unique specificity of a sialic acid binding lectin AchatininH, from the hemolymph of Achatina fulica snail

A sialic acid-binding lectin, AchatininH, from the hemolymph of Achatina fulica snail is found to be highly specific for 9-0-acetyl sialic acid. The binding specificity of AchatininH distinguishes it from other known sialic-acid specific lectins which usually show a broader range of specificity for sialic acid. It is even better than crab lectin which shows specificity for both 4- and 9-0-acetylated derivatives of sialic acid. This limited specificity of AchatininH appear to account for the fact that it agglutinates only rabbit, rat and guinea pig erythrocytes which contain 9-0-acetylated sialic acid but not horse (mainly contain 4-0-acetylated sialic acid), human, monkey, sheep, goat and chicken erythrocytes which contain either N-acetyl or N-glycolyl neuraminic acid but no 0-acetylated derivatives. This finding was further supported by the potent inhibition of hemagglutination by free 9-0-acetylated neuraminic acid and by several glyco shingolipids of human origin having 0-acetylated sialic acid.     

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.     

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.     

Mantle phenoloxidase activity and its role in sclerotization in a snail Achatina fulica

The precursors of sclerotin such as phenol and phenoloxidase have been localized in the mantle tissue of Achatina fulica based on incubation study. The phenolic compound exists as dopyl protein. The mantle shows both mono- and diphenoloxidase activity as evidenced by the effective oxidation of tyrosine and Dopa. The enzyme shows a pH optimum of 6.5. Chemicals acting on the mantle phenoloxidase reveal that it contains sulphydryl groups. Studies on the effect of temperature show that the heat inactivation of the enzyme is reversed at higher temperatures. Electrophoretic study indicates that the enzyme exists in multiple forms. Results are discussed in relation to sclerotization.     

Purification of achatin-I from the atria of the African giant snail, Achatina fulica, and its possible function.

Achatin-I previously purified from the ganglia of the African giant snail Achatina fulica was isolated from the atria of this snail. Achatin-I appeared to enhance the cardiac activity in two ways; centrally this peptide increased impulse frequency and produced spike broadening of the identified heart excitatory neuron, PON, and peripherally it enhanced amplitude and frequency of the heart beat. Achatin-I showed excitatory actions not only on the heart but on several other muscles.     

A novel cardio-excitatory peptide isolated from the atria of the African giant snail, Achatina fulica

An undecapeptide which potentiates the beat of the ventricle in the African giant snail, Achatina fulica Ferussac, was purified from the atria of the snail. Its primary structure was determined to be H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2. This peptide was found to have excitatory actions not only on the ventricle but also on the penis retractor muscle, the buccal muscle and the identified neurons controlling the buccal muscle movement of Achatina.     

Restoration of morphological and functional integrity in the regenerating eye of the giant African land snail Achatina fulica

To determine whether vision returns to its original state following eye removal in Achatina fulica, light and electron microscope examinations, electrophysiological recordings and behavioural tests were carried out on the regenerating snails. Reparative morphogenesis can result in the restoration of the peripheral sense organ even in the absence of complete regrowth of the tentacle, but it can also lead to the formation of aberrant regenerates. We found that anatomically and ultrastructurally the eyes of the ‘most normal’ regenerates were basically the same as the original eyes. Under normal conditions each eye is composed of a principal and an accessory eye, both sharing a common cornea. The only difference between regenerated and native eyes is the smaller size of the former, as a result of a reduced number of retinal cells. Electroretinographic responses revealed that the molecular mechanism of phototransduction is restored, in principle, but that flicker fusion frequency in the regenerated eye is significantly lower than in the normal eye. The directional movement to a visual stimulus (a black stripe of 45° width) had not completely recovered even 6 months after amputation. This suggests that the central projections of the optic nerve had not become fully re‐established at the time of testing.     

High molecular weight lectin isolated from the mucus of the giant African snail Achatina fulica

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca(2+)-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.     

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, Achatinin_H, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca^2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of Achatinin_H. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.