A 25-kDa Serine Peptidase with Keratinolytic Activity Secreted by <Emphasis Type="Italic">Coccidioides immitis</Emphasis>

Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.     

Protease activity in protein-free NSO myeloma cell cultures

Summary Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45–50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30–35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5–4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.     

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Preliminary analysis of the proteolytic enzymes in the excretory-secretory products of the adult stages of the dog hookworm Uncinaria stenocephala

The paper describes an introductory characterisation of proteinases present in the excretory-secretory products (ESP) of adult Uncinaria stenocephala. In SDS-PAGE gelatine substrate gels ESP resolved as a six bands of proteolytic activity, with a molecular weight of 182, 159, 98, 50, 39 and 26 kDa. The 98 and 39 kDa components were serine proteinases. The 50 kDa band was sensitive to a metalloproteinase inhibitor. The 26 kDa component was highly sensitive to cysteine proteinase inhibitors and was also partially inhibited in the presence of EDTA. The bands of 182 and 159 kDa were sensitive to a Zn-metalloproteinase inhibitor. The enzymes present in ESP showed the highest proteolytic activity at pH 8-9. Quantitative analysis revealed maximum proteolytic activity of the polypeptides of 159 and 182 kDa at pH 7; 98 and 26 kDa at pH 8 while the 50 kDa and 39 kDa components showed the highest activity at pH 9.     

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.     

Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D

Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Characterization of cathepsin L secreted by Sf21 insect cells

Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed.     

Extracellular Peptidase in the Fungal Pathogen Pseudallescheria boydii

Pseudallescheria boydii is a ubiquitous filamentous fungus capable of causing invasive disease in humans. In the present study, using sodium dodecyl sulfate–polyacrylamide gels containing bovine serum albumin as co-polymerized substrate, we identified a 28-kDa proteolytic activity released to the extracellular environment by mycelia of P. boydii. This peptidase was detected during the growth of P. boydii in Sabouraud-dextrose medium for 13 days and reached its maximal production on day 7. The 28-kDa peptidase was active in acidic pH (5.5) and had its activity completely blocked by 1,10-phenanthroline, a potent zinc-metallopeptidase inhibitor. Two other metallopeptidase inhibitors, EDTA and EGTA, were also tested and no alterations were observed in the activity of the 28-kDa extracellular peptidase. Likewise, E-64 (a cysteine peptidase inhibitor), phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), and pepstatin A (an aspartyl peptidase inhibitor) did not significantly alter the enzymatic behavior. Collectively, we described for the first time the expression of an extracellular metallopeptidase in the human opportunistic fungal pathogen P. boydii.     

Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.     

Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

We combined fluorogenic substrates or internally quenched fluorescent peptides with specific inhibitors in the pH profile of proteolytic activity experiments in order to detect proteolytic activities in lysates of MDCK cells. Hydrolytic activities related to cathepsin B, L, and D were observed. Serine-proteinase was not detected; however, we clearly demonstrated the presence of a thiol-metallo-endo-oligopeptidase, also called thimet-oligopeptidase (TOP). This peptidase from MDCK cells has substrate and inhibitor specificities as well as an activation profile with mercaptoethanol that are indistinguishable from the recombinant rat testis TOP (EC 3. 4.24.15). In addition, polyclonal purified antibodies to this enzyme depleted the TOP activity of MDCK cells in whole homogenate. Although we present only preliminary data, TOP is secreted by MDCK cells. The presence of TOP in a phenotype polarized MDCK cells can have special significance in the cytoplasmic selection, transport, or clearance of short peptides due to restriction of the enzyme to sequences from 6 to 17 amino acids. Therefore, the MDCK cell could be a very useful cellular model with which to study some of the suggested TOP biological functions as processing of biological active peptides and antigen presentation.     

Identification of cathepsin B,D and H in the larval midgut of colorado potato beetle,Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-Ala-Arg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.     

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.     

Purification and characterization of aspartic protease derived from Sf9 insect cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 μM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) μM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.     

Fasciola hepatica: A secreted cathepsin L-like proteinase cleaves host immunoglobulin

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatinsubstrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60–90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN₂, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60–90-kDa proteinases are cathepsin L-Iike proteinases.     

Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells

Testican-2 is a member of the testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was approximately 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit cathepsins L, B, K and S. We identified the C1q subcomponent of complement component C1 as a potential interacting partner of testican-2. The C1q subcomponent is a recognition molecule which acts in concert with other C1 subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes.