Hub genes and sub-networks of stoma-related genes in Hylocereus undatus through trypsin treatment during storage revealed by transcriptomic analysis.

To further investigate the preservation mechanisms of trypsin, the synergistic mechanisms of trypsin and stoma-related genes were evaluated in Hylocereus undatus. Trypsin significantly induced the stoma closure and improved the storage quality of H. undatus. Transcriptomic analyses of H. undatus revealed that important antioxidant signal pathway, such as SREBP signaling pathway, cellular response to H2 O2 or cellular response to molecule of bacterial origin, were induced; while responses to water deprivation were impeded by trypsin. These results indicated that trypsin relieved pitaya of pressure of water deprivation and exhibited the protection on pitaya during storage. Furthermore, the analyses of networks of protein-protein interaction suggested that OST1, HK5, AT4G27585, and HIR1 act as hubs of stoma-related proteins induced by trypsin during storage of H. undatus. PRACTICAL APPLICATIONS: Preservation of fruit is becoming increasingly important to the world. Keep the balance of production and scavenging of reactive oxygen species is efficient to improve the storage quality of fruit. Trypsin had a novel superoxide anion scavenging activity and protect fruit cells from cellular injury induced by excess ROS. This article investigates the hub genes and interaction mechanisms of stoma closure induced by trypsin during the storage of H. undatus. The application of trypsin provides a new strategy for the quality control of fruit storage. Trypsin will have a broad market and development potential in the area of food additives.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Effects of exogenous protease supplementation on endogenous trypsin activity and gene expression in broilers.

Two hundred and forty one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets with 0, 40, 80, or 160 mg/kg of an acid protease preparation from 1 to 42 days of age, in order to investigate the effects of an exogenous enzyme on growth performance, pancreatic trypsin activity and mRNA expression. Average daily gain (ADG) was greatest in the 160-mg/kg treatment group at 1-21 days, which was significantly higher than that in the control. After 42 days, average daily feed intake (ADFI) and ADG had significantly increased in the 80- and 160-mg/kg treatments (P < 0.05), with the 80-mg/kg treatment group having the highest ADFI and ADG values. The feed conversion ratio was not affected. Dietary acid protease at 80 and 160 mg/kg significantly decreased trypsin activity in the pancreas (relative to the control group) by 35.71 and 47.29%, respectively (P < 0.05). After 42 days, trypsin mRNA expression in the pancreas had significantly decreased by 19.5% in the 80-mg/kg treatment group relative to the control (P < 0.05). After 21 and 42 days, the diet supplemented with 160 mg/kg acid protease significantly decreased pancreatic trypsin mRNA by 19.6 and 37.7%, respectively, compared to the control. There were the 40-mg/kg treatment group and the control. Our results suggest that the amount of acid protease in the diet significantly affects trypsin activity and mRNA expression in broilers.

Characterization and functional classification of American lobster (Homarus americanus) immune factor transcripts.

The American lobster (Homarus americanus) is the most important commercially exploited marine species in Canada. Very little is known about the H. americanus molecular humoral immune response or how to determine if a seemingly healthy lobster is infected with a pathogen. The goal of this work is to characterize several important H. americanus immune genes as well as highlight and classify hundreds of others into functional immune groups. The protein sequence of H. americanus acute phase serum amyloid protein A (SAA) was found to be similar to that of vertebrate SAA, and is likely a good clinical marker for immune activation in lobsters and some crustaceans. Additionally, only one gene, Trypsin 1b, was found to be differentially regulated during bacterial, microparasitic and viral challenges in lobster and is likely critical for the activation of the H. americanus immune response. Bioinformatic analysis was used to functionally annotate, 263 H. americanus immune genes and identify the few shared patterns of differential gene expression in lobsters in response to bacterial, parasitic and viral challenge. Many of the described immune genes are biomarker candidates which could be used as clinical indicators for lobster health and disease. Biomarkers can facilitate early detection of pathogens, or anthropomorphic stressors, so that mitigation strategies can be developed in order to prevent the devastating economic losses that have occurred in Southern New England, USA. This work is contributes to further our understanding of how the lobster immune system works and how it can be used to maintain the health and sustainability of the overall American lobster fishery.

The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera.

The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.

A trypsin homolog in amphioxus: expression, enzymatic activity and evolution.

Trypsin has been documented in a variety of species including both vertebrates and invertebrates, but little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a trypsin gene in Branchiostoma japonicum. The cDNA was 978 bp long with an ORF encoding a deduced protein of 272 amino acids. The deduced protein had an N-terminal signal peptide of 15 amino acids, a 16 activation peptide with the typical cleavage site Arg/Ile, a Tryp_SPc domain with the catalytic triad His(72)-Asp(118)-Ser(215) and the S1 substrate binding residue Asp(209), which are all characteristic of trypsinogens. The recombinant trypsin protein was able to hydrolyse the trypsin prototypic substrate BAEE, which was inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. Both northern blotting and tissue-section in situ hybridization demonstrated that trypsin gene was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum, mid-gut and ovary. And the whole mount in situ hybridization showed that it began to express in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Phylogenetic analysis indicated that both amphioxus and ascidian trypsins are more closer to each other than to vertebrate trypsins, suggesting a continuous evolutionary divergence of vertebrate trypsins after split from protochordate/vertebrate common ancestor.

cDNA cloning, heterogeneous expression and biochemical characterization of a novel trypsin-like protease from Nilaparvata lugens.

A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to clone diverse trypsin-like protease gene transcripts from midguts of the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Six individual trypsin-like protease transcripts were identified. On the basis of one nucleotide sequence of the six clones, a full-length cDNA sequence (1902 bp) was obtained by rapid amplification of cDNA ends (RACE). The cDNA contained an 1128-bp open reading frame encoding a putative protein of 375 amino acids with typical features of the trypsin-like protease. Heterogeneous expression of the coding sequence for the mature peptide in Escherichia coli cells showed that the expressed protease with a molecular weight of 27.0 is active, for its BApNAse activity assayed by using BApNA (N-benzoyl-D,L-arginine-p-nitroanilide) as substrate. The protease had its maximum activity at pH 8.0 and 35 degrees C. A much better stability was observed at pH values above 4.0 and temperatures below 40 degrees C. The enzyme was strongly inhibited by serine protease inhibitor. The trypsin-like protease is therefore likely one of the major digestive proteases responsible for protein hydrolysis in N. lugens gut, and multiple gene families encoding digestive proteases may help in adaptation of this sap-sucker to different rice varieties.

Relationship of strain-dependent susceptibility to experimentally induced acute pancreatitis with regulation of Prss1 and Spink3 expression.

To analyze susceptibility to acute pancreatitis, five mouse strains including Japanese Fancy Mouse 1 (JF1), C57BL/6J, BALB/c, CBA/J, and C3H/HeJ were treated with either a cholecystokinin analog, cerulein, or a choline-deficient, ethionine-supplemented (CDE) diet. The severity of acute pancreatitis induced by cerulein was highest in C3H/HeJ and CBA/J, moderate in BALB/c, and mildest in C57BL/6J and JF1. Basal protein expression levels of the serine protease inhibitor, Kazal type 3 (Spink3) were higher in JF1 and C57BL/6J mice than those of the other three strains under normal feeding conditions. After treatment with cerulein, expression level of Spink3 increased remarkably in JF1 and mildly in C57BL/6J, BALB/c, CBA/J, and C3H/HeJ strains. Increased proteinase, serine, 1 (Prss1) protein expression accompanied by increased trypsin activity with cerulein treatment was observed in susceptible strains such as CBA/J and C3H/HeJ. Similar results were obtained with a CDE diet. In the 3 kb Spink3 promoter region, 92 or 8 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively, whereas in the Prss1 promoter region 39 or 46 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively. These results suggest that regulation of Prss1 and Spink3 expression is involved in the susceptibility to experimentally induced pancreatitis. The JF1 strain, which is derived from the Japanese wild mouse, will be useful to examine new mechanisms that may not be found in other laboratory mouse strains.

Low-barrier hydrogen bond hypothesis in the catalytic triad residue of serine proteases: correlation between structural rearrangement and chemical shifts in the acylation process.

To elucidate the catalytic advantage of the low-barrier hydrogen bond (LBHB), we analyze the hydrogen bonding network of the catalytic triad (His57-Asp102-Ser195) of serine protease trypsin, one of the best examples of the LBHB reaction mechanism. Especially, we focus on the correlation between the change of the chemical shifts and the structural rearrangement of the active site in the acylation process. To clarify LBHB, we evaluate the two complementary properties. First, we calculate the NMR chemical shifts of the imidazole ring of His57 by the gauge-including atomic orbital (GIAO) approach within the ab initio QM/MM framework. Second, the free energy profile of the proton transfer from His57 to Asp102 in the tetrahedral intermediate is obtained by ab initio QM/MM calculations combined with molecular dynamics free energy perturbation (MD-FEP) simulations. The present analyses reveal that the calculated shifts reasonably reproduce the observed values for (1)H chemical shift of H(epsilon)(1) and H(delta)(1) in His57. The (15)N and (13)C chemical shifts are also consistent with the experiments. It is also shown that the proton between His57 and Asp102 is localized at the His57 side. This largely downfield chemical shift is originated from the strong electrostatic interaction, not a covalent-like bonding character between His57 and Asp102. Also, it is proved that a slight downfield character of H(epsilon)(1) is originated from a electrostatic interaction between His57 and the backbone carbonyl group of Val213 and Ser214. These downfield chemical shifts are observed only when the tetrahedral intermediate is formed in the acylation process.

Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae).

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that approximately 26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae).

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.

Identification of novel serine proteinase gene transcripts in the midguts of two tropical insect pests, Scirpophaga incertulas (Wk.) and Helicoverpa armigera (Hb.).

We have used RT PCR and 3'RACE to identify diverse serine proteinase genes expressed in the midguts of the rice yellow stem borer (Scirpophaga incertulas) and Asian corn borer (Helicoverpa armigera). The RT-PCR primers encoded the conserved regions around the active site histidine57 and serine195 of Drosophila melanogaster alpha trypsin, including aspartate189 of the specificity pocket. These primers amplified three transcripts (SiP1-3) from midguts of S. incertulas, and two transcripts (HaP1-2) from midguts of H. armigera. The five RT PCR products were sequenced to permit design of gene-specific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of the 3'RACE products indicated that SiP1, SiP2 and HaP1 encoded trypsin-like serine proteinases, while HaP2 encoded a chymotrypsin-like serine proteinases. The SiP3 transcript proved to be an abundant 960 nt mRNA encoding a trypsin-like protein in which the active site serine195 was replaced by aspartate. The possible functions of this unusual protein are discussed.

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).

Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen.

Trypsin was purified from crayfish, Pacifastacus leniusculus, hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as a zymogen according to the sequence of the putative precursor peptide. The authenticity of the trypsinogen is supported by the deduced amino acid sequence and confirmed by the N-terminal amino acid sequence of the mature protein. The enzyme has features characteristic of a trypsin, such as a specific binding pocket. Sequence comparison shows that crayfish trypsin is similar to those of other species, with the exception that six cysteine residues present in vertebrates are missing. Some structural characteristics, such as the length of the signal peptide and a calcium binding site, are similar to bacterial trypsin.

Expression of acinar and ductal products in Capan-1 cells growing in synthetic serum and serum-free media.

BACKGROUND: Capan-1 is a human pancreatic adenocarcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum. METHODS: Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis. RESULTS: Capan-1, PANC-1, IBF-CP3, and MDAAmp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process. CONCLUSION: The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.

Correlation of haemagglutination activity with trypsin-like protease activity of Porphyromonas gingivalis.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with various forms of periodontal disease. Several characteristics of P. gingivalis are thought to contribute to its pathogenicity; these include haemagglutination and trypsin-like protease activity. Previous studies suggest an association between haemagglutination and trypsin-like protease activity of P. gingivalis. To investigate this, two complementary quantitative experimental approaches were taken. Five independent mutants of P. gingivalis deficient in trypsin-like protease activity were shown to exhibit reduced haemagglutination activity. In addition, enhancers (cysteine and dithiothreitol) and inhibitors (N-ethylmaleimide, N-p-tosyl-L-lysine-chloromethyl ketone, and phenylmethylsulphonyl fluoride) of trypsin-like protease activity were shown, respectively, to significantly enhance and inhibit haemagglutination activity of washed, wild-type P. gingivalis cells (p less than 0.05, paired t-test). Statistical analysis indicated a strong correlation between haemagglutination and trypsin-like protease activity (r = 0.85, p less than 0.001, Spearman rank correlation). The effect of the protease enhancers and inhibitors on haemagglutination activity was specific for P. gingivalis, as they did not significantly change the haemagglutination activity of Fusobacterium nucleatum. These results suggest that the proteolytic site of the trypsin-like protease participates in haemagglutination activity of P. gingivalis.