Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use.

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.

Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood.

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors, whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed, washed, immunomagnetically depleted of cells expressing glycophorin A and CD14, reacted for flow cytometric detection of ALDH, and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H, 10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed, banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid, myeloid and megakaryocytic blood elements.

Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei.

We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner. Antibody to native p67 identified a partial cDNA clone from a T. b. rhodesiense expression library and RT-PCR was used to complete the sequence of the cDNA. Equal levels of p67 transcript are detected in both procyclic and bloodstream stages of the life cycle. The 2771 nt cDNA contains a 1980 nt orf encoding a 659 amino acid polypeptide (72,567 Da). Hydropathy analysis predicts a Type I membrane topology (N to C): an N-terminal signal sequence, a large hydrophilic lumenal domain with 14 N-glycosylation sites, a trans-membrane domain (19 aa), and a short (24 aa) cytoplasmic domain. Peptide microsequencing of purified p67 identified nine residues identical to the deduced amino acid sequence, confirming the identity of the cDNA and defining the signal sequence cleavage site. Antibody to p67 protein produced in E. coli recognizes the same spectrum of native p67 glycoforms as the antibody used to clone the cDNA. All features of the deduced amino acid sequence are consistent with the known properties of the native protein and suggest a structure similar to mammalian LAMPS. The cytoplasmic domain contains two putative di-leucine targeting motifs similar to those involved in lysosomal targeting in vertebrate cells. Our results suggest that a single p67 polypeptide, or a group of highly related polypeptides, is synthesized in both bloodstream and procyclic trypanosomes and that subsequent post-translational processing and lysosomal targeting is subject to stage-specific regulation.

Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes.

We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.

Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes.

We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal compartments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37 degrees C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4 degrees C. When cells were incubated at 12 degrees C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12 degrees C. The morphology of collecting tubule membranes was specifically altered at 12 degrees C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12 degrees C were reversed 10 minutes after cells were returned to 37 degrees C. We also used flow cytofluorimetric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value considerably higher than that of mammalian lysosomes. Fluorescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when bloodstream forms were incubated in the presence of chloroquine.

Transport of a lysosomal membrane glycoprotein from the Golgi to endosomes and lysosomes via the cell surface in African trypanosomes.

gp57/42 is a membrane glycoprotein localized in the trans-Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans-Golgi. We refer to gp57/42 molecules that carry the CB1 epitope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diisocoumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37 degrees C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12 degrees C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket.(ABSTRACT TRUNCATED AT 400 WORDS)

Trypanosoma brucei brucei and T. b. gambiense: stumpy bloodstream forms express more CB1 epitope in endosomes and lysosomes than slender forms.

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.

Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote.

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

Trypanosoma brucei rhodesiense: membrane glycoproteins localized primarily in endosomes and lysosomes of bloodstream forms.

Bloodstream forms of Trypanosoma brucei rhodesiense take up macromolecules in endocytic vesicles that form in a large coated pit called the flagellar pocket. Glycoproteins that bind to ricin are concentrated in the flagellar pocket and in intracellular vesicles. We purified Triton X-100-soluble ricin-binding glycoproteins by lectin affinity chromatography and immunized mice to generate hybridomas. Monoclonal antibody produced by the CB1 hybridoma recognized heterodisperse trypanosome components migrating with M(r) 84-140 kDa in immunoblots. CB1 binding was specifically inhibited by lactose. The CB1-reactive material was purified by sequential affinity chromatography on ricin- and CB1-Sepharose. N-Glycosidase F, but not endoglycosidase H, digestion destroyed CB1-reactivity of purified material. This suggests that N-linked oligosaccharides contribute to the CB1 epitope. Glycosidase digestion of biosynthetically radiomethionine-labeled, affinity purified, CB1-reactive material yielded two radiolabeled polypeptides, p57 and p42. Thirteen methionyl peptides were resolved in one-dimensional peptide maps of V8 protease digests of p57; p42 had 10 methionyl peptides with mobilities indistinguishable from those of peptides of p57. This suggests that p57 and p42 are closely related. In cryoimmunoelectron microscopy studies CB1 specifically labeled the interior surface of tubular and vesicular membranes located between the nucleus and the flagellar pocket. These membranes were morphologically identical to structures that have been previously identified as trans Golgi, lysosomal, and endosomal elements. In double-labeling studies endocytosed serum albumen-gold complexes were found in the lumen of vesicles that had CB1-reactive material in their membranes. This provides direct evidence that vesicles containing high levels of CB1-reactive material are part of the lysosome/endosomal system. Some CB1-reactive material was also detected in the flagellar pocket by cryoimmunoelectron microscopy. Corrolated flow cytofluorimetry and immunofluorescence analysis showed that 85-96% of the total CB1-reactive material was intracellular and inaccessible to antibody in living cells. The 4-15% of the total CB1-reactive material accessible to antibody in living cells was localized in the flagellar pocket. Bloodstream forms of Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and T.b. rhodesiense all expressed the CB1 epitope. However, expression of this epitope is developmentally regulated during the parasite life cycle, for no CB1-reactive material was detected in procyclic forms. The trypanosome proteins detected by CB1 show some similarities to vertebrate lysosomal and endosomal membrane proteins.

Cholecystokinin cells purified by fluorescence-activated cell sorting respond to monitor peptide with an increase in intracellular calcium.

Cholecystokinin (CCK) is secreted from specific enteroendocrine cells of the upper small intestine upon ingestion of a meal. In addition to nutrients, endogenously produced factors appear to act within the gut lumen to stimulate CCK release. One such factor is a trypsin-sensitive CCK-releasing peptide found in pancreatic juice, known as monitor peptide. This peptide is active within the intestinal lumen and is hypothesized to stimulate CCK secretion by interacting directly with the CCK cell. We have found that monitor peptide releases CCK from isolated rat intestinal mucosal cells and that this effect is dependent upon extracellular calcium. In the present study, we used monitor peptide as a tool for isolating CCK cells from a population of small intestinal mucosal cells. Dispersed rat intestinal mucosal cells were loaded with the calcium-sensitive fluorochrome Indo-1, and CCK secretory cells were identified spectrofluorometrically by their change in fluorescence when stimulated with monitor peptide. Cells demonstrating a change in their emission fluorescence ratio were sorted using a fluorescence-activated cell sorter. More than 90% of the sorted cells stained positively for CCK with immunohistochemical staining. Furthermore, sorted cells secreted CCK when stimulated with membrane-depolarizing concentrations of potassium chloride, dibutyryl cAMP, calcium ionophore, and monitor peptide. These findings indicate that functional intestinal CCK cells can be highly enriched using fluorescence-activated cell sorting. Furthermore, monitor peptide appears to interact directly with CCK cells to signal CCK release through an increase in intracellular calcium.

Trypanosoma brucei rhodesiense bloodstream forms: surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone.

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.

A comparative study of D-lactate, L-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense.

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced D-lactate, but not L-lactate, whereas T. b. gambiense procyclic forms produced L-lactate, but not D-lactate, and T. lewisi produced both isomers.

Trypanosoma brucei sspp.: cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin.

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.

Trypanosoma brucei gambiense and T. b. rhodesiense: concanavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms.

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.

Flow cytofluorimetric analysis of drug accumulation by multidrug-resistant Trypanosoma brucei brucei and T. b. rhodesiense.

Dual laser flow cytofluorimetry has been used to compare accumulation of compounds representing three major classes of trypanocidal drugs by drug sensitive and drug resistant clones of Trypanosoma brucei brucei and T. b. rhodesiense. Clones selected for resistance to melarsoprol were shown to be cross-resistant in vivo to two diamidines, pentamidine and Berenil, but not to suramin. At 35 degrees C, bloodstream forms of these multidrug-resistant clones accumulated lower intracellular concentrations of the diamidines 4',6-diamidino-2-phenyl-indole (DAPI) and Hoechst 33342, the phenanthridine ethidium bromide, and the acridine acriflavine than drug sensitive parasites. Accumulation of all four drugs was saturable. Drug concentrations giving half-maximal rates of accumulation were increased in the resistant clones relative to the sensitive parent clones. The rate of DAPI accumulation by both resistant and sensitive parasites was strongly temperature dependent and increased sharply above 27 degrees C. Two distinct populations were resolved in mixtures of sensitive and resistant clones exposed to DAPI. Resistant and sensitive cells accumulated identical intracellular concentrations of DAPI following brief treatment with the detergent Triton X-100. The results suggest that alterations in the surface membrane of multidrug-resistant trypanosomes reduce accumulation of several drugs relative to drug sensitive parasites.

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides.

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma lewisi: restriction of alternative complement pathway C3/C5 convertase activity.

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment.

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.