The International Laboratory for Research on Animal Diseases (ILRAD) has recently developed interests in several areas of cell biology and biochemistry that are relevant to trypanosomiasis and theileriosis (East Coast fever [ECF]). The reasons for this interest stem from a perceived difficulty of tackling trypanosomiasis by conventional vaccination procedures due to the tremendous antigenic variation that trypanosomes can undergo. Thus, approaches other than a purely immunological one are now being explored. As a part of this new emphasis, a four-day workshop was held at ILRAD to review the current state of knowledge in the field of protein traffic and catabolism and to attempt to address these issues, with particular reference to parasitic diseases. The topics covered included such areas as protein targeting, protein assembly, protein degradation, endocytosis and lysosomal activity. There were 20 speakers from Europe and America and 8 from ILRAD, as well as approximately 40 observers from ILRAD and other research organizations in Africa. The organizers of this workshop would like to thank the United Nations Development Program (UNDP) for their financial support, and the distinguished visitors for their most valuable scientific contributions, which helped to make the meeting a success.
C. Redman (New York Blood Center) gave an elegant exposition on how a relatively simple procedure such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can be used to delineate the steps involved in such complex tasks as protein macromolecular assembly and disulphide cross-linking. The role of disulphide bond formation in protein assembly and targeting was also addressed by T. Morrison (University of Massachusetts Medical School) who showed that, while virion associated protein was exclusively in disulphide-linked oligomers, not all haemoglobin-nitrogen (HN) protein (in the Newcastle disease virus) was in this form during processing from the endoplasmic reticulum (ER). The importance of understanding such assembly process was exemplified by W. Fish (ILRAD), who showed that the immunological exposure of the species cross-reactive epitope, called the cross-reacting determinant (CRD), of the variant specific glycoprotein (VSG) of Trypanosoma congolense could be dissected into several distinct enzymatic and non-enzymatic (disulphide-bond reduction) steps. Apparently the enzymatic removal from VSG of the proposed membrane anchor, dimeristoylphosphatidyl-inositol, is not a pre-requisite for CRD exposure as formerly believed. Indeed, D. Grab (ILRAD) provided evidence that a phospholipase-C, which had been proposed to mediate the release of the surface coat from the parasite, was probably an intracellular enzyme, thus also raising doubts as to the proposed physiological role of the enzyme as a mediator for the release of VSG from the surface of the parasite.
The importance of correctly folded proteins was furthered explored by A. Goldberg (Harvard), who described the role of cytosolic proteases that degrade abnormally synthesized proteins and proteins that have been damaged by other processes such as the action of free radicals. Free radicals and their reaction products have long been recognized as agents capable of reacting with lipids and carbohydrates, but only recently was evidence found to show that proteins may also be affected. Interestingly, R. Dean (Brunei) showed that the bloodstream forms of African trypanosomes, which contain the thick VSG coating, are apparently more susceptible to free radical damage than are the same parasites from which the coat has been removed enzymatically, or those that have lost it (procyclic forms) in the normal course of the parasite life cycle! Such observations emphasize the importance of ascertaining the role of free radical damage to cellular proteins.
The purification and characterization of lysosomal from Entamoeaba histolytica (A. Barrett, Strangeways), and African trypanosomes (J. Lonsdale-Eccles, ILRAD) were described and their biochemical properties shown to be distinct from mammalian thiol-dependent cathepsins. The possibility that such differences between mammalian and parasite enzymes may be exploitable was addressed by E. Shaw (Freidrich Miescher Institute, Basel), who described a variety of peptidyl derivatives that acted as specific affinity-labelling inactivators of cysteine and serine proteinases. By appropriately tailoring the inhibitor, he was able to demonstrate the intracellular inhibition of different lysosomal enzymes as well as the blockage of intracellular processes such as antigen presentation or proinsulin processing. Interestingly, the trypanolytic activity of human serum could be blocked by lysosomotropic agents such as chloroquine (B. Betschart, Swiss Tropical Institute). J. Kay (University College, Cardiff) also described a modulation of intracellular antigen processing using an inhibitor that had a specificity against aspartyl proteases such as cathepsin E.
R. Kelly (University of California) described how sorting signals, which appear to be conserved in different cells, are involved in deflecting proteins from an otherwise default secretory pathway. K. Simons (European Molecular Biology Laboratory) addressed how such sorting can occur in polarized cells (e.g., Madin-Darby canine kidney cells, which can be infected by vesicular stomatitus virus only on the basolateral side and hepatitus A virus only on the apical side). He eliminated carbohydrate as a possible signal and showed that the lipid/glycolipid gradient from one side to the other is affected by a gate at the tight junctions between cells. By a novel technique (of decapitating the cells!) he was able to show that it is possible to dissect some of the biochemical events that are involved in such sorting. Whether trypanosomes, which are also polarized cells, will be amenable to such studies remains to be determined.
The biological importance of the lysosomal system was beautifully shown by E. Harms (Universitaets-Kinderklinik). He demonstrated the importance of several of the lysosomal enzymes using hereditary deficiencies of the enzymes to show their roles in the maintenance of the viability of the individual. A. Fok and R. Allen (Hawaii) gave detailed descriptions of the endocytotic pathways of Paramecium, whose endocytotic system is the best studied of the protozoans. Their digestive process appears to undergo a rapid, complex series of maturation events (approximately 30 minutes from internalization to defecation) that involves fusion of acidic vesicles and ultimate secretion of lysosomal contents. M. Moore (Plymouth, UK) described how lysosomes become destabilized when cells have been in contact with xenobiotics. This increased lysosome fragility is associated with enhanced catabolic activity. Destabilization of the lysosomal membranes can occur with a wide variety of perturbations, including pH. Indeed, using such properties, L. Huang (University of Tennessee) described the use of a pH-dependent drug delivery system that has been successfully employed in transfection and in other studies. Specific targeting of the liposomes is achieved by incorporating appropriate target antibodies in the surface of the liposome.
However, the use of immuno-liposomes will require a detailed knowledge of the intracellular compartments and sorting mechanisms. J. Slot (University of Utrecht), for example, has been studying how the lysosome membrane differs from that of the plasma membrane (he has proposed that sorting may be a consequence of changes in surface area within the organelles). A. Helenius (Yale University) has taken this one step further and suggested that there are two cycling systems, with early lysosomes and late lysosomes recycling their membranes in separate cycles. In the case of trypanosomes, progress in understanding sorting has been made by P. Webster (ILRAD). He has shown that there appears to be several sorting systems that may be effective according to either the chemical structure or the size of the respective compounds that are subject to sorting. This sorting appears to occur in a complex network of interconnected tubules or cisternae rather than in discete vesicles. In contrast, F. Maxfield (Columbia University) has shown that in mammalian CHO cells, 80% of a 2-macroglobulin goes to lysosomes whereas 90% of transferrin goes to the surface. Each appears to be in different-sized vesicles that are subject to different pH values in the different endocytic pathways. These endosomes fuse actively for several minutes after the initial internalization with a concomitant large increase in the number of non-recycling components but no increase in the recycled components (analogous to fractional distillation). To dissect the endocytotic steps, R. Anderson (University of Texas) has begun an ambitious project to study coated pit assembly in vitro. Initial studies suggest that he is able to prepare, in vitro, clathrin-coated pits that have properties similar to the in vivo ones.
During the blood-infective stage of their life cycle, T. brucei can undergo a morphologic differentiation that is accompanied by changes in several protein kinase substrates (Aboage-Kwarteng, ILRAD). These proteins are phosphorylated by cytosolic casein kinase-II-like enzymes that differ from mammalian casein kinase-II. Whereas the kinases from the African trypanosome appear to be involved in the switch from dividing to non-dividing cells, protein kinases in Theileria may act as growth signal transducers in the Theileria-induced-cell proliferation (O. ole-MoiYoi, ILRAD). The Theileria protozoan parasites are unusual in that they induce lymphoblastogenesis and clonal expansion of otherwise quiescent cells. A definitive role for the kinases in this transformation process has yet to be found. Although not causing transformation, plasmodial infection of cells causes morphologic changes in host structure. Some of these, M. Aikawa (Case Western Reserve University) reports, are to allow for the traffic of parasite protein to the host cell surface.
