Variant-specific Surface Proteins Research Articles

Purification of a Variant-specific Surface Protein of Giardia lamblia and Characterization of its Metal-binding Properties

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

Giardia lamblia trophozoites contain multiple alleles of a variant-specific surface protein gene with 105-base pair tandem repeats

Giardia lamblia trophozoites undergo antigenic variation of a variant-specific surface protein (VSP). All VSPs that have been reported have had high cysteine contents, including numerous copies of a CXXC motif. The first vsp gene described (vspA6; from the cloned line, WBA6), contained 21 copics of a 195 base pair tandem repeat, but other reported VSPs have not contained repeats. In this report, we describe the vsp gene from WBC5, a cloned line derived from WBA6. The vspC5 gene contains short 5′ and 3′ regions flanking 26 copies of a 105-bp tandem repeat, which comprises 93% of the coding region. In addition to the copy containing 26 repeats, the genome contains other copies of the vspC5 with fewer copies of the repeat. The sequences flanking the repeats are identical, and all copies map to the same location on chromosomal Band 5. suggesting that multiple alleles of the vspC5 gene are present.

Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number.

Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units.

Allele-specific expression of a variant-specific surface protein (VSP) of Giardia lamblia.

The surfaces of Giardia lamblia trophozoites demonstrate variable expression of a set of cysteine-rich surface proteins, called variant-specific surface proteins (VSP). The cloned Giardia line, WBA6, expresses a 170 kD VSP (VSPA6 or CRP170) which contains approximately 18 to 23 copies of a 65 amino acid repeat. We have cloned the expressed vspA6 gene containing 23 repeats from a genomic library as well as copies of the vspA6 gene with only 8 or 9 repeats from both WBA6 and from WB1269, a cloned line derived from WBA6 which has lost the expressed copy of the gene. The recombinant clones containing the genes with only 8 or 9 repeats have 8 nucleotide substitutions in the coding region. All the recombinant clones map to the same chromosomal location, yet RNA sequencing and comparison with the transcript size indicate that only the clone with 23 repeats contains a gene producing a stable transcript. The most likely interpretation of these data is that G.lamblia trophozoites contain multiple alleles of the vspA6 gene of which only one is expressed.

Variant-specific surface proteins of Giardia lamblia are zinc-binding proteins.

Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) are a distinct family of cysteine-rich proteins. Characteristically, cysteine residues occur mostly as CXXC tetrapeptides. Four of the reported five VSPs contain a putative metal-binding domain that resembles other metal-binding motifs; the fifth is closely related but lacks an essential histidine. Three different native VSPs bound Zn2+. Co2+, Cu2+, and Cd2+ inhibited Zn2+ binding. Analysis of recombinant VSP fusion proteins showed that the putative binding motif bound Zn2+. Surprisingly, peptide fragments from other regions of the VSP contain numerous CXXCXnCXXC motifs that also bound Zn2+. Analysis of deduced amino acid sequences showed well-conserved CXXC spacing in three out of five VSPs, suggesting conservation of structure despite amino acid sequence divergence. The function of VSPs is unknown, but by binding Zn2+ or other metals in the intestine, VSPs may contribute to Zn2+ malnutrition or inhibition of metal-dependent intestinal enzymes, which would lead to malabsorption, a well-known consequence of giardiasis.

Identification and characterization of a Giardia lamblia group-specific gene

Giardia lamblia consist of heterogeneous isolates that can be divided into at least three groups. Differential screening of a cDNA library with isolate-specific antisera identified a gene which is expressed and found only in Group 3 isolates. This gene, GLORF-C4, is 597 bp in length and predicts a deduced protein of 198 amino acids that is characterized by a polyserine motif. Giardia can also be grouped by their ability to express certain variant-specific surface proteins (VSPs), expression of which is restricted among groups. In Southern blots, probes specific to two VSPs were used to characterize isolates. Failure to detect VSP genes correlated with inability to express the same VSP. Analysis of isolates with these new probes complements and confirms the groupings previously suggested using other criteria. These genetic differences should allow differentiation of isolates and permit the application of basic epidemiological techniques to determine the manner of spread and the presence of animal reservoirs.

Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size

Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) of isolate WB are cysteine-rich, can vary dramatically in size, contain Cys-X-X-Cys motifs, and are differentially expressed. GS/M(H7) is a Giardia clone from a different isolate which expresses a VSP epitope not found in WB. The VSP gene encoding this epitope was selected by differential hybridization using radiolabeled cDNA from H7 and variant sibling trophozoite lines from the same isolate that express other VSPs. The VSPH7 gene probe detects and 1800 nucleotide transcript abundant in H7 but undetectable in variant siblings. Primer extension directly from RNA was used to complete the gene sequence which predicted a protein with a molecular weight of 56 832. The protein showed many of the characteristics of 2 previously sequenced WB VSPs including many Cys-X-X-Cys tetrapeptides and a conserved carboxy-terminal region. Genomic Southern analysis indicated the presence of 2 distinct VSPH7 genes in H7. An oligonucleotide from the conserved region was used in combination with one specific for the VSPH7 gene to estimate the VSP repertoire size at between 133 and 151. VSPs, even from isolates expressing unique epitopes, constitute a family of related proteins.

Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia

Antigenic variation in the parasitic protozoan Giardia lamblia was studied by characterizing the expression and genomic organization of a variant-specific surface protein (VSP) gene. Transcripts from this gene, vsp1267, were abundant in the cloned variant WB/1267, but undetectable in the parental clone from which WB/1267 was derived or in variant progeny of WB/1267. Two identical copies of vsp1267 exist in the WB/1267 genome, separated by 3 kb and arranged as convergent transcription units. Primer extension sequencing and S1 nuclease protection analysis suggested that the 5′ untranslated region (UTR) of VSP1267 mRNA consists of a single nucleotide (nt). Primer extension sequencing mapped the site of VSP1267 transcript polyadenlyation 25 nt beyond the termination codon. vsp1267 contained no introns and predicted a cysteine-rich polypeptide with features common to other VSPs. Comparison of vsp1267 with another VSP gene sequence revealed striking conservation, both at the nucleotide and amino acid levels, at the 3′ ends of the genes. An oligonucleotide derived from this region detected size-variant VSP transcripts in 4 of 5 G. lamblia clones analyzed, suggesting the general utility of this probe in studying VSP genes and their expression.