Alpha-helical coiled-coil structures of Trypanosoma brucei variable surface glycoproteins

We have used electron microscopy to examine purified intact variable surface glycoproteins (VSGs) from clones derived from two distinct stocks of Trypanosoma brucei. The VSG molecule from MITat 1.2 has a large elongated domain consistent with the shape of the dimeric N-terminal domain determined by X-ray analysis (see preceding paper), and a heretofore unseen short, thin fibrous tail presumed to be the C-terminal domain. Electron microscopy on DiTat 1.3, however, indicates a morphology quite distinct from that of MITat 1.2. Analysis of four VSG amino acid sequences reveals 7-fold periodicities (heptad repeats) which indicate that alpha-helical coiled-coil secondary structure elements occur in all of these VSGs, consistent with the observation of helical bundles in one VSG. These results suggest the possibility that VSG antigenic diversity may be related to a diversity in length and disposition of alpha-helical bundles and coiled-coil domains.     

6 A-resolution X-ray structure of a variable surface glycoprotein from Trypanosoma brucei

The variable surface glycoprotein (VSG) is the predominant component of the surface coat of the African trypanosome. The expression of antigenically distinct VSGs on minor populations during infection allows the parasite to escape the host immune response. Purification of the protein is facilitated by the enzymatic release of a soluble form of VSG (sVSG) which occurs on cell lysis. The soluble form is a dimer with an approximate molecular weight of 120,000-130,000. Partial proteolysis of sVSG reveals a protease-sensitive link between an amino-terminal domain which comprises about two-thirds of the molecule, and a C-terminal domain which contains the membrane attachment site. We have obtained crystals suitable for high-resolution structural analysis from preparations of three sVSG: MITat 1.2, ILTat 1.25 and ILTat 1.22. The crystal structure of the dimer of the MITat 1.2 amino-terminal domain has been solved to 6 A resolution. We report here that the dimer is an unusual 90 A rod-like molecule composed of a helical bundle of at least four 80 A-long alpha-helices.     

Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei

Kinetoplastids are unicellular eukaryotes that include important parasites of man, such as trypanosomes and leishmanias. The study of these organisms received a recent boost from the development of transient transformation allowing the short-term expression of genes reintroduced into parasites like Trypanosoma brucei, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants of T. brucei that have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neo(r) gene) into the trypanosome tubulin cluster. Plasmids in which part of the T. brucei tubulin gene cluster has been replaced by the neo(r) gene were used. Targeting efficiency was higher with a linearized than with a circular construct, and with 5 kilobases of tubulin gene cluster than with 0.9 kilobases. With these neo(r) constructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.     

Stability of expression-linked surface antigen gene in Trypanosoma equiperdum

African trypanosomes evade clearance in immune-competent hosts by periodically replacing their major surface glycoprotein with an antigenically different glycoprotein. Expression of many of these variant surface glycoproteins (VSGs) is associated with the duplication and transposition of silent basic copy genes (BCs) into unlinked genomic expression sites. The new expression-linked VSG gene copies (ELCs) are oriented with their 3' ends proximal to chromosome telomeres. Other VSG genes are activated without the production of an ELC. The 3' ends of these VSG genes are near chromosome telomeres both when they are active and when they are inactive. Recently, we have shown that activation of the VSG-1 gene in the BoTaR (Bordeaux trypanozoon antigen repertoire) serodeme of Trypanosoma equiperdum involves the duplication and transposition of a telomeric BC gene into one of at least three unlinked telomeric sites. Here we show that the VSG-1 ELC is inactivated but not eliminated in some antigenic variants derived from a VSG-1 expressor. In addition, a subsequent variant that again expresses VSG-1 has not reactivated the residual VSG-1 ELC (R-ELC), but instead contains a new, active VSG-1 ELC in an unlinked telomeric site. These results show that the simple presence of an ELC in a potential expression site is not sufficient for its expression.     

Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei

The protozoan parasite Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly (Glossina spp.). Trypanosomes ingested by the fly undergo a number of changes in the insect midgut during differentiation to procyclic forms. These include the loss of the variant specific glycoprotein (VSG) coat and the appearance of a common set of procyclic surface antigens. In order to investigate genes other than VSG genes which are expressed only at certain stages of the life cycle, the first cDNA specific to procyclic culture form trypanosomes (equivalent to the stage found in the insect midgut) has been characterized. The encoded polypeptide shows several characteristics of membrane proteins, but its most striking feature is the presence of a repetitive amino-acid sequence in which there are 22 tandem repeats of the dipeptide-Glu-Pro-. Related genes are also found in other trypanosome species and in leishmania. This gene shows many similarities to a number of surface antigen genes described in malaria and, more recently, Trypanosoma cruzi. This is the first example of a repetitive sequence in a parasite protein which is present only in the insect vector, and which therefore cannot be implicated in the mammalian host immune response.     

Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum

African trypanosomes are thought to evade the host immune system by periodically changing their variable surface glycoprotein (VSG). VSG genes are activated by a complex process involving the duplicative transposition of silent basic copy genes to one of several expression sites. These expression-linked copies (ELCs) of the VSG genes are also subject to regulation within expression sites by as yet unknown mechanisms. It is generally assumed that trypanosomes can express only one VSG gene at a time. Nevertheless, the finding that they contain multiple VSG gene expression sites suggests that multiple expression is possible. We show here that Trypanosoma equiperdum can stably express two VSG genes in a simple axenic culture system and that both antigens are present on the cell surface. The two antigens do not co-cap or form heterodimers. Their corresponding genes show no cross-hybridization and are situated in different telomere-linked expression sites. Northern blot analysis reveals that both genes are active in the double expressors.     

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.     

Two variant surface glycoproteins of Trypanosoma brucei of different sequence classes have similar 6 A resolution X-ray structures

Antigenic variation in the African trypanosome is mediated through changes in the composition of the surface coat. By controlling expression of the major surface protein, the variant surface glycoprotein (VSG), from a repertoire of perhaps 1,000 different genes the organisms exhibit a series of antigenically distinct coats and evade the host's immune system. We have determined the 6 A resolution structure of a T. brucei variant surface glycoprotein, ILTat 1.24, using X-ray crystallography. The crystallized protein consists of the N-terminal two-thirds of the intact VSG which has a relative molecular mass (Mr) of 60,000 (60K). The structure, which includes a 90 A long alpha-helical bundle, is strikingly similar to that of the N-terminal fragment of VSG MITat 1.2 (ref. 4). Although most known VSG sequences show little similarity of primary sequence in the N-terminal domain, the similarity between the structure of a Class I (ILTat 1.24) and a Class II (MITat 1.2) VSG antigen suggests that VSGs may share a common tertiary structure.     

一类亚循环2-群自同构群的阶及机器实现

首先研究一类亚循环2-群的自同构群的阶。其基本方法是根据所给亚循环表示(不一定是标准表示)中的生成元关系,对其自同构进行机械式运算,计算出自同构群的阶及相关子群的阶。然后,在机械式运算的基础上,通过C语言进行编程,实现其自同构群的阶的机器计算。其机器运算的结果表明,亚循环群及其自同构群的机器证明和机器计算是可行的。     

Role of self-peptides in positively selecting the T-cell repertoire

The fate of an immature thymocyte is determined by the specificity of its alpha beta T-cell receptor. Only cells expressing receptors that interact with sufficient affinity with major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and go on to mature and seed the peripheral lymphoid organs. The H-2Kb class-I MHC molecule positively selects for the maturation of cytotoxic T lymphocytes that will respond in the periphery to H-2Kb cells presenting a foreign peptide. We have now analysed the ability of variant H-2Kb molecules to positively select T-cells that respond to H-2Kb with ovalbumin. Our results indicate that self-peptides, presented in the groove of the class-I molecule on thymus epithelial cells, are critically involved in positive selection of the T-cell repertoire. Furthermore, the ability of four different H-2Kb variants to select this response in the thymus correlates with their ability to present the ovalbumin peptide, indicating that a self-peptide mimic of the foreign peptide could be involved in positive selection.