Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.

In order to promote an efficient humoral immune response, germinal center B cells modify both the antigen recognition and effector domains by programmed genetic alterations of their antibody genes. To do so, B cells use the enzyme activation-induced deaminase (AID), which transforms deoxycytidine into deoxyuridine at the immunoglobulin genes, triggering mutagenic DNA repair. Data accumulated during the past decade have significantly advanced our understanding of how AID activity is regulated and preferentially targeted to the immunoglobulin genes. There is also a better understanding of the ways by which AID-catalyzed uracil is recognized and the ensuing downstream processing underpinning the mechanisms of somatic hypermutation and class switch recombination. Here, we critically review these advances in the context of their relevance for the humoral immune response. A detailed understanding of these molecular mechanisms is paramount to uncover the basis of B cell intrinsic immunodeficiency, as well as to suggest tools and strategies that might allow boosting antibody gene diversification in the context of immunizations or infections that require the elicitation of rare or highly mutated antibody variants.

The protozoan Trypanosoma cruzi has a family of genes resembling the mucin genes of mammalian cells.

Mucins are heavily O-glycosylated Thr/Ser/Pro-rich molecules. Given their relevant functions, mucins and their genes have been mainly studied in higher eukaryotes. In the protozoan parasite Trypanosoma cruzi, mucin-like glycoproteins were shown to play an important role in the interaction with the surface of the mammalian cell during the invasion process. We show now that this parasite has a family of putative mucin genes, whose organization resembles the one present in mammalian cells. Different parasite isolates have different sets of genes, as defined by their central domain. Central domains, rich in codons for Thr and/or Ser and Pro residues, are made up of either a variable number of repeat units in tandem or non-repetitive sequences. Conversely, 5'- and 3'-ends from different genes in different isolates have similar sequences, suggesting their common origin. Comparison of deduced amino acid sequences revealed that all members of the family have the same putative signal peptide on the N terminus and a putative sequence for glycophosphatidylinositol anchoring on the C terminus. The deduced molecular mass of the core proteins is small (from 17 to 21 kDa), in agreement with the 1-kilobase size of the mRNA detected. Putative mucin genes in T. cruzi are located on large chromosomal bands of about 1.6-2.2 megabase pairs.

AU-rich elements in the 3'-untranslated region of a new mucin-type gene family of Trypanosoma cruzi confers mRNA instability and modulates translation efficiency.

Trypanosoma cruzi has a complex mucin gene family of 500 members with hypervariable regions expressed preferentially in vertebrate associated stages of the parasite. In this work, a novel mucin-type gene family is reported, composed of two groups of genes organized in independent tandems and having very short open reading frames. The structures of deduced proteins share the N and C termini but differ in central regions. One group has repeats with the consensus Lys-Asn-Thr(7)-Ser-Thr(3)-Ser(Ser/Lys)-Ala-Pro and the other a Thr-rich sequence of the type Asp-Gln-Thr(17-20)-Asn-Ala-Pro-Ala-Lys-Asp-Thr(5-7)-Asn-Ala-Pro-Ala-L ys. In both cases, expected mature core proteins are around 7 kDa. Both groups, named L and S, respectively, differ in the structure of genomic loci and mRNA, with differential blocks in the 3'-untranslated region. The highest mRNA level for S and L groups are in the epimastigote stage but they show distinct developmentally regulated patterns. Transcripts are short lived and their steady-state abundance is regulated post-transcriptionally with increased mRNA stability in insect stage epimastigote. AU-rich sequences, similar to ARE motives known to cause mRNA instability in higher eukaryotes, are present in the 3'-untranslated region of the transcripts. In transfection experiments this sequence is shown to be functional for the L group destabilizing its mRNA in a stage-specific manner. Furthermore, an effect of this AU-rich region on translation efficiency is shown. To our knowledge, this is the first time that a functional ARE sequence-dependent post-transcriptional regulation mechanism is reported in a lower eukaryote.

The Trypanosoma cruzi mucin family is transcribed from hundreds of genes having hypervariable regions.

In previous works we have identified genes in the protozoan parasite Trypanosoma cruzi whose structure resemble those of mammalian mucin genes. Indirect evidence suggested that these genes might encode the core protein of parasite mucins, glycoproteins that were proposed to be involved in the interaction with, and invasion of, mammalian host cells. We now show that the mucin gene family from T. cruzi is much larger and diverse than expected. A minimal number of 484 mucin genes per haploid genome is calculated for a parasite clone. Most, if not all, genes are transcribed, as deduced from cDNA analysis. Comparison of the cDNA sequences showed evidences of a high mutation rate in localized regions of the genes. Sequence conservation among members of the family is much higher in the untranslated (UTR) regions than in the sequences encoding the mature mucin core protein. Transcription units can be classified into two main subfamilies according to the sequence homologies in the 5'-UTR, whereas the 3'-UTR is highly conserved in all clones analyzed. The common origin of members of this gene family as well as their relationships can be defined by sequence comparison of different domains in the transcription units. The regions encoding the N and C termini, supposed to correspond to the leader peptide and membrane-anchoring signal, respectively, (Di Noia, J. M., Sánchez, D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149) are highly conserved. Conversely, the central regions are highly variable. These regions encode the target sites for O-glycosylation and are made of a variable number of repetitive units rich in Thr and Pro residues or are nonrepetitive but still rich in Thr/Ser and Pro residues. The region putatively coding for the N-terminal domain of the mature core protein is hypervariable, being different in most of the transcripts sequenced. Nonrepetitive central domains are unique to each gene. Gene-specific probes show that the relative abundance of different mRNAs varies greatly within the same parasite clone.

High diversity in mucin genes and mucin molecules in Trypanosoma cruzi.

Mucins are highly O-glycosylated molecules which in mammalian cells accomplish essential functions, like cytoprotection and cell-cell interactions. In the protozoan parasite Trypanosoma cruzi, mucin-related glycoproteins have been shown to play a relevant role in the interaction with and invasion of host cells. We have previously reported a family of mucin-like genes in T. cruzi whose overall structure resembled that of mammalian mucin genes. We have now analyzed the relationship between these genes and mucin proteins. A monoclonal antibody specific for a mucin sugar epitope and a polyclonal serum directed to peptide epitopes in a MUC gene-encoded recombinant protein, detected identical bands in three out of seven strains of T. cruzi. Immunoprecipitation experiments confirmed these results. When expressed in eukaryotic cells, the MUC gene product is post-translationally modified, most likely, through extensive O-glycosylation. Gene sequencing showed that the central domains encoding the repeated sequences with the consensus T8KP2, varies in number from 1 to 10, and the number of Thr residues in each repeat could be 7, 8, or 10. A run of 16 to 18 Thr residues was present in some, but not all, MUC gene-derived sequences. Direct compositional analysis of mucin core proteins showed that Thr residues are much more frequent than Ser residues. The same fact occurs in MUC gene-derived protein sequences. Molecular mass determinations of the 35-kDa glycoproteins further extend the heterogeneity of the family to the natural mucin molecules. Difficulties in assigning each of the several MUC genes identified to a mucin product arise from the high diversity and partial sequence conservation of the members of this family.

Trypanosoma cruzi surface mucins with exposed variant epitopes.

The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.