Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining beta-elimination and fixed-charge derivatization.

A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The beta-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44 Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues.

Studies of acceptor site specificities for three members of UDP-GalNAc:N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC.

The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP) containing clusters of threonine residues mimicking the mucin tandem repeat unit of MUC5AC. The O-glycosylated products obtained after in vitro reactions were fractionated by capillary electrophoresis and the purified glycopeptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalNAc residues was transferred into the MUC5AC motif peptide and the preferential order of incorporation for each GaNTase isoform was determined. Our results suggest that clusters of threonine appear to be essential for site recognition of peptide backbone by the ubiquitous GaNTases and also support the notion that the different GaNTase isoforms with varying substrate specificities are involved in a hierarchical order of O-glycosylation processing of the mucin-type O-glycoproteins.

Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides.

The recombinant proteins of the two novel UDP-N-acetylgalactosamine (GalNAc) glycopeptide:N-acetylgalactosaminyltransferases (designated gpGaNTase-T7 and gpGaNTase-T9) were assayed with O-glycosylated products obtained from the prior action of the ubiquitous transferases (GaNTase-T1 and GaNTase-T2) towards MUC5AC mucin motif peptides (GTTPSPVPTTSTTSAP and peptides with single amino acid substitutions, GTTPSAVPTTSTTSVP and GTTPSPVPTTSITSVP, that are a reflection of mucin molecule polymorphism). gpGaNTase-T9 is known to be expressed differentially and more abundantly than gpGaNTase-T7 in some tissues; the results of in vitro glycosylation also indicates a difference in acceptor substrate specificities between the gpGaNTase isoforms. With the use of capillary electrophoresis, MS and Edman degradation, our study suggests that, in the O-glycosylation of mucin-type proteins, approach and recognition signalling by gpGaNTase-T7 and gpGaNTase-T9 depend largely on the peptide's primary structure (for example the presence of multiple clusters of hydroxy amino acids and the number of GalNAc residues attached to the peptide backbone). O-glycosylation in terms of sites of attachment seems to be less random than previously described and, if sequential reactions are ordered throughout the Golgi stack, the complete O-glycosylation of the mucin molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.

Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9.

We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.

Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity.

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide.

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.

O-glycosylation potential of lepidopteran insect cell lines.

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.

Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry.

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.

Dipeptidyl aminotransferase activity and in vitro O-glycosylation of MUC5AC mucin motif peptides by human gastric microsomal preparations.

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.

Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s).

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamineto human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

O-Glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line.

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performing in vitro reactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype. As acceptor for the UDP-N-acetylgalactosamine:polypeptide Nacetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used. A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different pattern of galactosaminylation in pre- and postconfluent cells. Core 1 UDP-galactose:N-acetyl-alpha-galactosaminyl-R 3-beta-galactosyltransferase (3-beta-galactosyltransferase) activityalso decreased with the differentiation, whereas CMP-neuraminic acid:galactose-beta-1, 3-N-acetyl-alpha-galac- tosaminyl-R 3-alpha-sialyltransferase activity increased. In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-beta-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an alpha-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation.

Mucin gene expression in the human endocervix.

In the human uterine endocervix, five out of the six human mucin genes investigated (MUC 2, MUC 3, MUC 4, MUC 5AC, MUC 5B and MUC 6) are expressed, whereas in some other mucosae the hybridization pattern demonstrates the expression of only two or three of these genes. The most intense labelling by in-situ hybridization is obtained significantly with MUC 4, predominantly during the luteal phase. The expression of the MUC 4 gene appears to be influenced by the oestro-progesterone ratio. During the ovulatory cycle, there are only a few differences concerning the variations of expression of all other MUC genes.

Analysis by electrospray mass spectrometry of glycopeptides from the in vitro O-glycosylation reaction using human mucin motif peptide.

A mucin-motif peptide in the one-letter code T T T P S P P M T T P I T P P A, representative of the human intestinal mucin tandem repeat sequence (MUC2), containing several threonine residues in clusters, was used as an acceptor substrate to investigate the effect of peptide structure on the activity of crude preparation of human gastric UDP-GalNAc:polypeptide N-acetyl galactosaminyltransferases. High-performance liquid chromatography was performed to separate the different products of the in vitro O-glycosylated reaction. The electrospray mass spectrometry was used to identify the different masses (m/z) of these products. Although the m/z of glycopeptide(s) could be higher than the detection limits of the spectrometer, an accurate study of the doubly charged ions allowed us to demonstrate the linkage of more than two sugars. Hence, the peptide MUC2 will accept at least four carbohydrate residues but the exact substituted positions should be confirmed by further sequence determination.

Combination of high-performance anion-exchange chromatography and electrospray mass spectrometry for analysis of the in vitro O-glycosylated mucin motif peptide.

Reversed-phase high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection were developed for the study of products obtained from the in vitro O-glycosylation of a mucin motif peptide, TTSAPTTS, the most representative sequence encoded by the human gene MUC5C. After incubation of the peptide, which is rich in clustered hydroxyamino acids, by both human colonic and gastric microsomal homogenates, the glycosylated products were separated by HPLC and HPAEC and analysed by electrospray mass spectrometry (ES-MS). The combination of HPAEC and ES-MS was the approach used for evaluating the differences between the polypeptide N-acetylgalactosaminyltransferase activity in different digestive tissues.

Hypogonadism induced by African trypanosomes in humans and animals.

1. The clinical syndromes of the African trypanosomiasis (also called sleeping sickness in humans) are defined. The basic knowledge on the hypothalamo-anterior pituitary-gonad axis functions is briefly recalled. 2. Hypogonadism caused by the trypanosomes both in men and in women as well as in male and female animals are extensively reviewed in publications over the last two decades as well as on our own very recent works which provided new insights on the physiopathology of the gonadal disorders.

Unusual cleavage of peptidic hormones generated by trypanosome enzymes released in infested rat serum.

Gonad and thyroid dysfunctions are often observed in human and experimental models during african trypanosomiasis. The enzymatic activity of components released by the trypanosomes towards peptide hormones (e.g. GnRH, TRH) have consequently been studied. The incubation products of GnRH by (i) healthy or infested rat serum: (ii) trypanosomal components released by using a specific procedure; (iii) infested and normal rat brain extracts have been analysed by RP-HPLC fractionation. The peptide cleavage has been assessed by determination of either the amino acid compositions or relative molecular weight (by FAB mass spectrometry) of the different resolved HPLC fractions. Different protease inhibitors and a reducing agent have also been tested and a serine, cation-sensitive, thiol-dependent endopeptidase activity has been predominantly identified to be released by the trypanosomes in host circulation. It has been shown that the peptidase activity(ies) is(are) able to: (i) degrade the peptide hormones (GnRH, TRH) considered as important neuromodulators and neurotransmitters; (ii) generate an unusual N-terminal tetrapeptide (GnRH1-4) appearing to be still active towards the gonadal hypothalamo-pituitary axis.

Experimental "chronic" African trypanosomiasis: endocrine dysfunctions generated by parasitic components released during the tryptanolytic phase in rats.

The disorders of the gonadotropic axis have been studied during the course of a "chronic" african trypanosomiasis induced experimentally in rats inoculated by the variant Trypanosoma brucei brucei AnTat 1.1.E. The levels of serum and pituitary LH as well as serum testosterone and corticosterone have been determined, during the infestation, at a particular period of the circadian cycle, in regard to the parasitemia variations. In addition, the inoculation of trypanosomal component fractions [obtained by concanavalin-A sepharose chromatography (conA-components)], has been performed in an attempt to define more exactly the nature of factor(s) producing the hypotestosteronemia in rats. This work evidenced that the hormonal parameter levels were predominantly decreased at the trypanolytic phase during the evolution of the disease. The action towards the hypothalamo-pituitary gonadal axis was attributed not only to peculiar trypanosomal enzyme(s) [a serine, thiol-dependent, cation sensitive endoprotease with a post-proline cleaving activity (purified from unretained conA fraction)], but also to protein and/or glycoprotein factor(s) released by the trypanosomes (components with affinity to the lectin).

Decrease of testosterone level during an experimental African trypanosomiasis: involvement of a testicular LH receptor desensitization.

To investigate gonadal disorders and changes of the testicular receptors occurring during the sleeping sickness disease (African trypanosomiasis), an experimental model was developed with 10-month-old rats infested by bloodstream forms of two variants of Trypanosoma brucei brucei (AnTat 1.1 A and AnTat 1.8). At the acute phase, three days after inoculation, the animals were sacrificed for estimating the serum levels of LH and testosterone and the number of testicular LH receptors. Considering a possible intervention of the stress during the infestation and to improve our investigations on gonadal imbalance related to trypanosomasis, levels of additional parameters [corticosterone, glucose and transaminases (glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase)] were determined. Stimulation testing with hCG was likewise assessed in infested rats to analyse the testicular testosterone response to gonadotropin. A significant decrease was demonstrated for serum LH and testosterone levels in the infested rats, as well as the loss of: (i) the testicular responsiveness to exogenous gonadotropin; (ii) the number of testicular LH receptors. Moreover, the remaining testicular receptors of infested rats showed an increase in their equilibrium association constant (Ka). Our study suggests that dysfunction of Leydig cells occurring during African trypanosomiasis is in part related to stress induced by the presence of the parasites.

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.