Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB³H₄ labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB³H₄ labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.The tsetse fly-transmitted protozoan parasite Trypanosoma brucei causes human sleeping sickness and the cattle disease Nagana in sub-Saharan Africa. The organism undergoes a complex life cycle between the mammalian host and the insect, tsetse, vector. The bloodstream form of the parasite expresses a dense monolayer of glycosylphosphatidylinositol (GPI)- anchored variant surface glycoprotein dimers and avoids specific immune responses through antigenic variation (32, 47). Following ingestion in a blood meal, the parasites differentiate into procyclic-form parasites that colonize the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat that includes about 3 × 10⁶ procyclin glycoproteins (28, 36, 37) and about 1 × 10⁶ poly-N-acetyllactosamine-containing free GPIs (19, 29, 39, 55). The procyclins are polyanionic, rod-like (38, 50) proteins encoded by procyclin genes. In T. brucei strain 427, used in this study, the parasites contain (per diploid genome) two copies of the GPEET1 gene, encoding 6 Gly-Pro-Glu-Glu-Thr repeats; one copy each of the EP1-1 and EP1-2 genes, encoding EP1 procyclins with 30 and 25 Glu-Pro repeats, respectively; two copies of the EP2-1 gene, encoding EP2 procyclin with 25 Glu-Pro repeats; and two copies of the EP3-1 gene, encoding EP3 procyclin with 22 Glu-Pro repeats (1). The EP1 and EP3 procyclins contain a single N-glycosylation site, occupied exclusively by a conventional Man₅GlcNAc₂ oligosaccharide, at the N-terminal side of the Glu-Pro repeat domain (1, 50). Whereas neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on six out of seven Thr residues (25). In culture, the procyclin expression profile depends on the carbon source (56) and metabolic state of the cells (27), and in the tsetse fly, there appears to be a program of procyclin expression such that GPEET procyclin is expressed early, giving way to EP1 and EP3 procyclin expression (2, 54). GPEET and EP procyclins contain similar GPI membrane anchors. These are based on the ubiquitous ethanolamine-P-6Manα1-2Manα1-6Manα1-4GlcNα1-6PI core (where, in this case, the PI lipid is a 2-O-acyl-myo-inositol-1-P-sn-2-lyso-1-O-acylglycerol structure 50]), but they also contain the largest and most complex known GPI side chains. These side chains are large poly-disperse-branched poly-N-acetyllactosamine structures (with an average of about 8 to 12 repeats, depending on the preparation) that can terminate with α2- and α3-linked sialic acid residues (9, 50). Sialic acid is transferred from serum sialoglycoconjugates to terminal β-galactosidase residues by the action of a cell surface GPI-anchored trans-sialidase enzyme (7, 26, 34). The trans-sialylation of surface components plays a role in the successful colonization of the tsetse fly (29). In vivo, the N termini of the procyclins are removed by tsetse fly gut proteases (2), though the role of this event is unclear (20) and it is thought that the underlying (protease-resistant) anionic repeat units and associated GPI anchor side chains might protect the parasite from the approach of tsetse fly gut hydrolases (2).The cell surface architecture of procyclic trypanosomes has been manipulated by the gene knockout of the procyclin genes themselves (55, 57), by galactose starvation (39), and by the knockout or knockdown of genes encoding enzymes of the GPI biosynthetic pathway, i.e., TbGPI10, TbGPI8, and TbGPI12 (11, 19, 29, 30). The procyclin TbGPI10 and TbGPI8 knockouts all resulted in parasites devoid of GPI-anchored procyclins, but this was compensated for by an upregulation in free GPI expression. However, the TbGPI12 null mutants that cannot synthesize GPI structures beyond GlcNAc-PI, revealed the presence of previously unidentified non-GPI-anchored surface coat components. In this paper, we characterize the fate of non-GPI-anchored procyclin protein and characterize the non-GPI-anchored surface coat components.