High Molecular Weight Protease in Rumen Ciliate Protozoa

Nagasawa, Takashi; Nagase, Tatsuo; Onodera, Ryoji

doi:10.1271/bbb.58.2020

Cited by 7 publications

(5 citation statements)

References 9 publications

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“…Trichomonas [24, 281, Trypanosomes [29], Dictyostelium [ 11. Acanthamoeba [30], Tetrahymena [31] and rumen ciliates [32]. The secretion of these cysteine proteases by protozoan parasites is thought to be responsible for specific host-parasite interactions related to their virulence ; attempts have been made to use these enzymes as potential targets for therapeutic intervention (for review see [3]).…”

Section: Discussionmentioning

confidence: 99%

Cathepsin L is an Intracellular and Extracellular Protease in Paramecium Tetraurelia

Völkel

Kurz

Under

et al. 1996

European Journal of Biochemistry

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The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDWPAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatik characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His, tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 pM.Keywords: cathepsin L; propeptide; expression ; Paramecium tetraurelia.Proteases serve many distinct functions in the cellular metabolism. Intracellularly, most obvious roles are in degradation of ingested food and turnover of cellular proteins. This usually occurs in lysosomes or digestive vacuoles. Other functions exist in the developmental and metabolic control of specific proteins through activation or inactivation by limited proteolysis [ 11. Extracellularly, secreted proteases are used to access solid proteinacous food or to facilitate tissue invasion via collagenolytic and elastinolytic activities as reported for some pathogenic bacteria, parasitic protozoa, and ras-transformed tumor cells [2-41. In the unicellular ciliate Paramecium tetraurelia the formation and processing of digestive vacuoles and lysosomes has been investigated predominantly by electron microscopy [5]. In these studies, 16 hydrolases of presumed lysosomal localization have been superficially identified more than a decade ago. Among those were the proteolytic cathepsins B, C, and D [ 5 ] . Nothing is known about extracellular proteases. During attempts to identify and study protein phosphatases from P. tetraurelia, we noticed the presence of a highly active protease in cell homogenates that hydrolyzed the '*P-labelled phosphorylase a which was used as a substrate for the protein phosphatases, to smaller ['*P]phosphopeptides. These labelled peptides were not acid-precipitable and thus quenched the phosphatase assay. Because hitherto no detailed enzymatic study of any protease from this ciliate had been reported, we decided to examine the nature of this protease activity. After separation from protein phosphatase two prot...

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Section: Discussionmentioning

confidence: 99%

Cathepsin L is an Intracellular and Extracellular Protease in Paramecium Tetraurelia

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Kurz

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et al. 1996

European Journal of Biochemistry

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“…Broderick et al (1988) indicated that free peptides originating from dietary protein degradation accumulate in the rumen, becoming available for protozoal attack. In the presence of protozoa, because of active protozoal peptidases (Newbold et al, 1989) and proteases (Naga and el-Shazly, 1968;Coleman, 1983;Forsberg et al, 1984;Nagasawa et al, 1994), dietary proteins and peptides are more susceptible to cleavage, which will release free AA into the medium. Moreover, Coleman (1975) proposed that protozoa utilize only about onehalf of their ingested N, the rest being expelled as shortchain peptides and free AA.…”

Section: Methodsmentioning

confidence: 99%

Effects of lauric acid on ruminal protozoal numbers and fermentation pattern and milk production in lactating dairy cows1

Faciola

Broderick

Hristov

et al. 2013

Journal of Animal Science

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The objectives of this study were to evaluate lauric acid (LA) as a practical ruminal protozoa-suppressing agent and assess effects of protozoal suppression on fermentation patterns and milk production in dairy cows. In a pilot study, 6 lactating Holstein cows fitted with ruminal cannulae were used in a randomized complete-block design trial. Cows were fed a basal total mixed ration (TMR) containing (DM basis) 15% alfalfa silage, 40% corn silage, 30% rolled high moisture shelled corn, and 14% solvent soybean meal, and assigned to 1 of 3 treatments: 1) control, 2) 160 g/d of LA, or 3) 222 g/d of sodium laurate, which is equimolar to 160 g/d of LA, all given as a single dose into the rumen via cannulae before feeding. Both agents showed high antiprotozoal activity when pulse dosed at these amounts via ruminal cannulae, reducing protozoa by 90% (P<0.01) within 2 d of treatment. Lauric acid reduced ruminal ammonia concentration by 60% (P<0.01) without altering DMI. Both agents reduced ruminal total free AA concentration (P<0.01) and LA did not affect ruminal pH or total VFA concentration. In a large follow-up feeding trial, 52 Holstein cows (8 with ruminal cannulae) were used in a randomized complete-block design trial. Cows were assigned to 1 of 4 diets and fed only that diet throughout the study. The TMR contained (DM basis) 29% alfalfa silage, 36% corn silage, 14% rolled high moisture shelled corn, and 8% solvent soybean meal. The 4 experimental diets were similar, except part of the finely ground dry corn was replaced with LA in stepwise increments from 0 to 0.97% of dietary DM, which provided (as consumed) 0, 83, 164, and 243 g/d of LA. Adding these amounts of LA to the TMR did not affect DMI, ruminal pH, or other ruminal traits, and milk production. However, LA consumed at 164 and 243 g/d in the TMR reduced the protozoal population by only 25% and 30% (P=0.05), respectively, showing that these levels, when added to the TMR, were not sufficient to achieve a concentration within the rumen that promoted the antiprotozoal effect of LA.

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“…Moreover, Broderick et al (1988) indicated that free peptides originating from dietary protein degradation might accumulate in the rumen and be available for protozoal attack, which might contribute to free TAA accumulation in the rumen. In the presence of RP because of active protozoal proteases (Naga and el-Shazly, 1968;Coleman, 1983;Forsberg et al, 1984;Nagasawa et al, 1994) and peptidases (Newbold et al, 1989), dietary proteins and peptides are more susceptible to cleavage, which will release free AA into the medium. Coleman (1975) proposed that RP utilize only about onehalf of their ingested N; the rest are expelled as shortchain peptides and free TAA.…”

Section: Methodsmentioning

confidence: 99%

Effects of feeding lauric acid on ruminal protozoa numbers, fermentation, and digestion and on milk production in dairy cows1

Faciola

Broderick

2013

Journal of Animal Science

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The objectives of this study were 1) to determine the level of lauric acid (LA) addition to the diet necessary to effectively suppress ruminal protozoa (RP) to the extent observed when a single dose was given directly into the rumen, 2) to assess LA effects on production and ruminal metabolism, and 3) to determine the time needed for RP to reestablish themselves after LA is withdrawn from the diet of lactating dairy cows. In Exp. 1, 2 Holstein cows fitted with ruminal cannulae were used in a split-plot design pilot study. Both cows consumed the same level of LA, starting with 0 g/d and increasing to 129, 270, and 438 g/d mixed into the diet. Diets were fed as total mixed ration (TMR) and contained (DM basis) 30% corn silage, 30% alfalfa silage, and 40% concentrate. Lauric acid intake linearly decreased DMI (P = 0.03), RP numbers (P < 0.01), ruminal acetate molar proportion (P = 0.03), and ruminal ammonia concentration (P = 0.03). Lauric acid intake linearly increased ruminal valerate molar proportion (P = 0.02). A quadratic response of LA consumption was observed on total ruminal VFA concentration (P < 0.01) and propionate molar proportion (P < 0.01), with maximum responses at 270 g/d of LA intake. A quadratic response of LA consumption was also observed on total ruminal free amino acid (TAA) concentration (P < 0.01), with minimum concentration at 270 g/d of LA intake. After withdrawing the greatest LA dose from the diet, RP returned to their original numbers in 12 d. In Exp. 2, 48 multiparous Holstein cows (8 with ruminal cannulae) were blocked by days in milk into 12 blocks of 4 cows (2 blocks of cannulated cows) and randomly assigned within replicated 4 × 4 Latin squares to balanced dietary treatment sequences. Diets were fed as TMR and contained (DM basis) 36% corn silage, 29% alfalfa silage, and 35% concentrate, and LA intake levels were 0, 220, 404, and 543 g/d mixed in the TMR. In Exp. 2, LA linearly reduced RP (P < 0.01), ruminal ammonia (P < 0.01), and total free AA concentration (P < 0.01); however, dietary LA also linearly decreased DM intake (P < 0.01). Intake of LA linearly reduced ruminal total VFA concentration (P < 0.01); DM, OM, NDF, and CP digestibility (P < 0.01); and milk production and milk components (P < 0.01). Therefore, LA does not appear to be a feasible RP suppressant for feeding in practical diets.

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High Molecular Weight Protease in Rumen Ciliate Protozoa

Cited by 7 publications

References 9 publications

Cathepsin L is an Intracellular and Extracellular Protease in Paramecium Tetraurelia

Cathepsin L is an Intracellular and Extracellular Protease in Paramecium Tetraurelia

Effects of lauric acid on ruminal protozoal numbers and fermentation pattern and milk production in lactating dairy cows1

Effects of feeding lauric acid on ruminal protozoa numbers, fermentation, and digestion and on milk production in dairy cows1

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