Partial Structure of Glutamic Acid and Alanine-rich Protein, a Major Surface Glycoprotein of the Insect Stages of Trypanosoma congolense *

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenusNanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenusTrypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the major surface glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing ansn-1-stearoyl-2-l-3-HPO4-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growingin vitro.

The life cycle of the African trypanosome alternates between a bloodstream stage in the mammalian host and insect stages in the tsetse. The parasite is adapted to survive in these very different host environments by producing upon entering its new environment distinct protective surface coats of glycosylphosphatidylinositol (GPI) 1 -anchored glycoprotein. The bloodstream form trypanosome is covered with variant surface glycoprotein (VSG), and the large repertoire of VSG genes allows the parasite to evade the mammalian immune system by antigenic variation (1)(2)(3). In the tsetse, the trypanosome differentiates to the procyclic form, sheds its VSG coat and produces a new layer of glycoprotein. In Trypanosoma brucei, the procyclic and epimastigote stage glycoproteins belong to the procyclin family composed mainly of repeat sequences (4 -6). There are two types of procyclin termed "EP" and "GPEET," which differ in their repeat sequences (7)(8)(9). The procyclins provide a highly acidic protease-resistant coat that is proposed to help the parasite survive in the harsh environment of the tsetse fly gut. It has recently been shown that the non-repetitive N-terminal domains of T. brucei procyclins are cleaved during exposure to the tsetse, whereas the majority of the protein remains stable (10), consistent with the idea that the procyclins function to protect the parasite surface from digestive enzymes. Extensive structural characterization of the procyclins has revealed an unusually large and complex GPI anchor (7,8,11), which may also be important for parasite survival. The phosphatidylinositol (PI) portion of the GPI anchor contains a L-glycerolipid and is acylated on the 2-hydroxyl of the inositol ring. The latter feature makes it resistant to the action of phospholipase C, PI-phospholipase C (7).
In Trypanosoma congolense, the corresponding procyclic surface coat is comparatively uncharacterized. In 1993, two groups (12,13) independently described the identification of a major surface antigen on procyclic T. congolense. A gene encoding this protein was cloned by differential screening of a procyclic T. congolense cDNA library and named GARP for glutamate and alanine-rich protein (12). GARP is one of at least three major surface glycoconjugates in T. congolense (14), and it contains no repeat sequences and shares no homology with T. brucei procyclins. However, the molecules are thought to be functionally equivalent because they share stage specificity, surface disposition, and immunodominance. Furthermore, the introduction of GARP into procyclin-deficient T. brucei restored the ability of the null mutant to establish heavy infection in the tsetse mid-gut (15).
Southern analysis of DNA from one strain of T. congolense has shown that multiple GARP genes exist at two distinct loci (16). At the first locus, GARP1, there is a single gene (gA), whereas locus GARP2 exhibits allelic variation with ten GARP genes at one allele and six at the other allele. The sequences of six of the GARP2 genes (gB1-6) have been reported (16) and are highly conserved (Fig. 1).
Immunogold electron microscopy has shown dense packing of GARP on the T. congolense cell surface (12), but little is known regarding the structure of the glycoprotein and its GPI anchor.
Here, we set out to purify and characterize T. congolense GARP. Using a combination of mass spectrometry techniques along with enzymatic and chemical treatments, we have determined the partial structure of GARP.

Cell Culture
T. congolense TREU 1457 procyclic/epimastigote cells were grown in minimum Eagle's medium (Invitrogen) supplemented with 15% fetal bovine serum and 10% serum plus (JRH Bioscience) at 27°C. Cultures were maintained by diluting into fresh medium, ensuring a concentration of a least 1 ϫ 10 6 cells/ml Ϫ1 .

Purification of Glycoconjugates
T. congolense (10 10 cells) were washed in ice-cold phosphate-buffered saline. The pellet was extracted twice with 10 ml of chloroform/methanol/water (1:2:0.8 v/v). After centrifugation, the pellet was dried under a stream of N 2 and extracted twice in 10 ml of 5% 1-propanol in 100 mM ammonium acetate (Solvent A). The extract was centrifuged (15700 ϫ g, 10 min) and loaded at 5 ml/h Ϫ1 onto an octyl-Sepharose column (15 ϫ 1 cm) preequilibrated in the same buffer. The column was washed with 20 ml of Solvent A and eluted at 10 ml/h Ϫ1 with a 120-ml linear gradient from Solvent A to 60% 1-propanol in water. Fractions (1 ml) were collected. To separate GARP from co-eluting glycolipids, peak fractions were applied to a longer octyl-Sepharose column (35 ϫ 1 cm) and eluted with a 300-ml linear gradient from 10% 1-propanol in 100 mM NH 4 Ac to 40% 1-propanol. The separation of GARP from co-eluting glycolipids was followed by dot blotting as described below. Small-scale octyl-Sepharose chromatography of a tryptic digest of GARP was performed on a 1-ml column eluted with a 40-ml gradient from Solvent A to 60% 1-propanol.

Dot Blot Analysis of Column Fractions
Aliquots (1 l) of the octyl-Sepharose fractions were spotted onto nitrocellulose membrane and blocked for 1 h at room temperature with blocking buffer (3% (w/v) bovine serum albumin in phosphate-buffered saline containing 0.1% (v/v) Tween 20). Membranes were then probed with either anti-GARP rabbit polyclonal antibody directed against the GARP peptide sequence or a mouse monoclonal antibody directed against an unknown carbohydrate epitope (12). Primary antibodies were diluted at 1:5000 in blocking buffer and incubated for 1 h at room temperature. Membranes were then washed three times in wash buffer (phosphate-buffered saline containing 0.1% (v/v) Tween 20) for 5 min for each wash. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Scottish Antibody Production Unit, Lanarkshire, United Kingdom) were diluted 1:5000 in wash buffer and incubated for 1 h at room temperature before washing as described above. The immunoprobed dots were detected using ECL-Plus detection reagents (Amersham Biosciences) according to the manufacturer's instruction.

SDS-PAGE and Western Blot Analysis
Samples of GARP and glycoinositol phospholipids were resolved on polyacrylamide mini-gels (12-15%) and electrophoretically transferred to nitrocellulose or PVDF membrane (Amersham Biosciences) using a semi-dry blot apparatus (Hoefer) at 20 mA/gel. GARP and glycoinositol phospholipids bound to the membrane were detected as described above for the immunoprobing of dot blots.

Periodate Silver Staining of SDS-PAGE Gels
GARP-containing SDS-PAGE gels were stained with periodate silver according to a previously described method (17,18). Gels were fixed overnight in ethanol, acetic acid, and water (40:5:55 v/v) and then oxidized in 0.7% (w/v) sodium periodate dissolved in fixing solution for 15 min with gentle agitation. Gels were then washed three times in 500 of ml water before incubating in silver nitrate solution, which was made fresh by adding 1 ml of 0.2 g/ml Ϫ1 AgNO 3 dropwise to a mixture of 5.6 ml of 0.1 N NaOH and 0.4 ml of c.NH 4 O, diluting with 17 ml of water and mixing gently for 15 min. Gels were then washed three times in 500 ml of water and developed with 200 ml of 0.05 mg/ml Ϫ1 citric acid, 0.1% (v/v) formaldehyde.

Enzymatic and Chemical Cleavages
Mild Acid Hydrolysis-GARP was treated with 40 mM trifluoroacetic acid for 10 min at 100°C. After hydrolysis, samples were dried in a SpeedVac and resuspended in water.
Aqueous Hydrogen Fluoride (aq.HF) Dephosphorylation-Dried samples were treated with 50 l of 48% aq.HF at 0°C for 48 h before freeze-drying to remove the aq.HF. Samples were then dried twice from 50 l of water to remove residual HF.
Tryptic Digests-For in-gel digests, GARP was localized by staining with Sypro Ruby and processed according to a method described previously (19). For off-blot tryptic digests, GARP was localized by immunostaining of adjacent lanes with anti-GARP antibodies. The unstained GARP-containing nitrocellulose band was cut into 1-mm 2 pieces, and sufficient 1% n-octyl glucoside in 20 mM NH 4 HCO 3 was added to cover the nitrocellulose pieces. The samples were incubated for 20 min at room temperature before trypsin (sequencing grade, Roche Molecular Biochemicals) was added to a final concentration of 30 -50 g/ml Ϫ1 . The samples were incubated overnight at 30°C on a shaking platform.

Off-blot Release and Extraction of GARP PI
A 500-pmol aliquot of purified GARP was run on SDS-PAGE and Western blotted onto PVDF membrane. The band containing GARP and a negative control band with no protein were excised and deaminated with 800 l of 0.3 M sodium acetate, pH 4.0, and 800 l of fresh 1 M NaNO 2 at 37°C for 3 h. The PVDF membrane was washed three times with 0.5 ml of water and then extracted three times with 0.5 ml of water-saturated butan-1-ol with vortexing for 20 min followed by 10 min of sonication. The combined butanol extracts were dried, resuspended in 100 l of chloroform/methanol 4:1 (v/v), and applied to a small (100 -200 l) Silica gel (Si60, Merck) column prewashed with 10 volumes of chloroform/methanol 4:1 (v/v). The column was then washed with 20 volumes of chloroform/methanol 4:1 (v/v), and the PIs were subsequently eluted in 10 volumes of chloroform/methanol 1:4 (v/v) for analysis by electrospray-ionization mass spectrometry (ES-MS).

ES-MS and ES-MS-MS
Negative ion electrospray mass spectra were recorded on either a Micromass Quattro Ultima triple quadrupole instrument or a Micromass Q-Tof2 instrument. Samples were infused in chloroform/methanol 1:4 at either 5 l/min Ϫ1 (Quattro Ultima) or using a nanospray tip (Q-Tof2). The capillary/tip and cone voltages were 2.7 kV and 50 V (Quattro Ultima) or 900 V and 40 V (Q-Tof2). Daughter ion ES-MS-MS spectra were recorded on the Q-Tof2 with a collision energy of 45 V.

Matrix-assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry
Positive ion MALDI-TOF mass spectra of GARP were acquired on an ABI Voyager DE-STR instrument in linear mode using sinapinic acid as the matrix. Tryptic digests of GARP (aliquots of 0. 5 l) were mixed 1:1 with ␣-cyano-4-hydroxycinnamic acid matrix for analysis by positive ion reflectron mode, MALDI-TOF.

Composition Analysis
The carbohydrate and inositol contents of purified GARP were determined from GARP, transferred to PVDF membrane by SDS-PAGE, and Western blotted using fresh transfer buffers. The GARP band was excised from the PVDF membrane along with bands directly above and below the GARP band as negative controls. The PVDF strips were placed in 2-ml glass Reacti-Vials (Pierce) containing 250 l of 4 M trifluoroacetic acid in 25% methanol and incubated at 100°C for 4 h. The PVDF strips were removed, and the hydrolysates were dried in a SpeedVac and redried twice from 50 l of water to remove residual trifluoroacetic acid. The products were redissolved in 100 l of water containing [1,2,3,4,5,6-2 H]myo-inositol and scyllo-inositol internal standards, and samples were transferred to capillary tubes for standard inositol and monosaccharide analyses by gas chromatography-mass spectrometry (GC-MS) (20).

RESULTS AND DISCUSSION
GARP Is a Highly Glycosylated GPI-anchored Glycoprotein-Purified GARP was analyzed by SDS-PAGE. GARP does not stain well with conventional protein stains like Coomassie Blue, Sypro Orange, or Sypro Ruby, but it stains intensely with periodate silver, a sensitive carbohydrate and protein stain. The GARP preparation appeared as a single diffuse band with an apparent molecular mass of 40 -44 kDa (Fig. 2A, lane 1). This apparent molecular mass is significantly higher than those predicted (ϳ21.6 kDa) from the GARP gene sequences (Fig. 1). This discrepancy suggested a high level of post-translational modification such as glycosylation. To identify the true mass of GARP, the purified sample was analyzed by MALDI-TOF mass spectrometry, which showed that the molecule was polydisperse with an average mass of ϳ30 kDa (data not shown). Inositol analysis by GC-MS demonstrated the presence of significant amounts of myo-inositol as expected for a GPIanchored molecule, and monosaccharide analysis by GC-MS revealed 24 Man and 18 Gal residues/inositol. On average, these 42 hexose residues/molecule would account for ϳ6.8 kDa (23% by weight) of the glycoprotein. The presence of contaminating glycolipids in the GARP preparation that might skew the inositol and monosaccharide composition can be ruled out from the periodate silver-staining pattern in Fig. 2A, lane 1), which shows that apart from some faint keratin bands above the GARP band, there is a complete absence of low molecular weight carbohydrate-containing material. A standard of 1 g of Trypanosoma cruzi epimastigote glycoinositol phospholipids (21) is shown alongside (Fig. 2A, lane 2) to illustrate this point. In addition, the off-blot method of composition analysis used in this study further reduces the danger of contamination. The yield of GARP based on inositol content was 3 nmol/10 10 cells, suggesting a minimum copy number of 2 ϫ 10 5 copies/cell. Eventhough this figure does not allow for losses during purification, it is significantly lower than the 2 ϫ 10 6 copies of procyclin/cell on T. brucei procyclic-formed cells. This finding is consistent with the recent observation that GARP is only one of at least three major cell surface molecules in T. congolense insect-stage parasites (14).
To account for the carbohydrate content, the GARP peptide sequences were analyzed for potential glycosylation sites. None of the GARP sequences contains any NX(S/T) N-glycosylation sites, but using an algorithm developed for mammalian Oglycosylation (22)(23)(24), two potential O-glycosylation sites were found in all of the GARP sequences at Thr-89 and Thr-159 (Fig.  1). In addition, it was considered possible that GARP might contain a highly substituted GPI anchor similar to that of T. brucei procyclin (7, 11).

Localization of the Carbohydrate Constituents of GARP-
Experiments were performed to distinguish between O-glycosylation and/or GPI substitution. When GARP was dephosphorylated with ice-cold 48% aq.HF for 48 h, a large shift in apparent molecular mass from ϳ44 to 32 kDa was seen by Western blot analysis using an antiserum against the GARP polypeptide (Fig. 2B, lane 2). MALDI-TOF-MS analysis of the sample treated for 16 h with aq.HF showed that the 30-kDa polydisperse material had been modified to polydisperse material of ϳ26 kDa and much less disperse material of ϳ21.6 kDa (Fig. 3A). Further aq.HF treatment for a total of 48 h led to the conversion of more sample to the terminal 21.6-kDa product (Fig. 3B). The 21.6-kDa figure is consistent with the predicted amino acid sequences of the seven reported GARP gene sequences, assuming that the mature proteins start at Gln-14, the preferred N terminus predicted by the signal peptide algorithm (25), and end at Asp-221, one of the three potential GPI additional sites predicted by the Big PI Predictor algorithm (26,27). However, we cannot exclude the possibility that the GARP  (16) were aligned using ClustalW. The underlined sequences at the N and C termini are those predicted to be N-terminal signal sequences and C-terminal GPI additional signal peptides by SignalP (25) and Big PI Predictor (26,27), respectively. The aspartic acid residues in bold represent alternative possible GPI addition sites. The one chosen in the figure is based on the MALDI-TOF results in Fig. 3 compared with the predicted molecular weights of the protein components plus ethanolamine (EtN) shown following each sequence. The bold and underlined Thr residues are those predicted to be modified by NetOGlyc (22)(23)(24). proteins have GPI added to Asp-215 or Asp-218 and are processed elsewhere at the N terminus and/or contain a posttranslational modification that is resistant to aq.HF.
The Western blot and MALDI-TOF results with the aq.HFtreated material could be interpreted as meaning that there are no direct O-linked glycans on GARP and that all of the carbohydrate is associated with the GPI anchor, such that it is lost upon scission of the ethanolamine phosphate bridge of the GPI anchor as was described for procyclin (28,29). However, this model does not readily explain the formation of the 26-kDa intermediate. Furthermore, a similar result was observed when the sample was treated with mild acid (40 mM trifluoro-  acetic acid, 100°C, 10 min) (Fig. 2B, lane 3). This treatment cleaves only extremely acid-labile linkages such as Asp-Pro peptide bonds not present in any of the GARP sequences and sugar-1-phosphate bonds. The latter are found in oligosaccharide-P-Thr/Ser linkages in a number of lower eukaryotes (30) including the Leishmania (31, 32), T. cruzi (33), and possibly T. brucei (34) and also in GPI anchor side chains of the unrelated protozoans Paramecium (35). Thus, the reduction in apparent molecular mass by mild acid treatment suggests that all or most of the carbohydrate of GARP is linked via phosphodiester bonds to Ser/Thr residues and/or to the core glycan of the GPI anchor. To address the latter possibility, a sample of purified GARP was digested with trypsin and then applied to a small octyl-Sepharose column. The unbound peptides/glycopeptides were washed through the column, and the bound Cterminal GPI-peptide was eluted with a 1-propanol gradient. An analysis of the GPI-peptide fraction for inositol and monosaccharides by GC-MS revealed a composition of Gal:Man: inositol of ϳ5:2:1. Because the non-reducing terminal Man residue is cryptic to conventional GC-MS monosaccharide analysis as a result of the stability of the Man-6-P phosphate group (36), we may conclude a composition of Gal:Man:inositol of ϳ5:3:1. The Man 3 Ins 1 composition is typical of the minimal GPI anchor core. The presence of ϳ5 Gal residues/GPI anchor is reminiscent of the Gal side chains found on the GPI anchors of T. brucei bloodstream form of VSGs (36,37). Unfortunately, a lack of material prevented further characterization of this fraction. However, it would appear that the majority of the Man and Gal of GARP is not associated with the GPI anchor but is presumably attached by phosphodiester linkage to the GARP polypeptide.
Taking into account the discrete 26-kDa intermediate, the prediction of two O-glycosylation sites, and the composition of the GPI anchor, we suggest that there are two oligosaccharide chains attached via phosphate to Thr-89 and Thr-159 of average composition Man 11 Gal 6.5.
MALDI-TOF Analysis of GARP Tryptic Digests-Mass fingerprint analysis of tryptic digests of native and chemically modified GARP indicated that more than one GARP gene is expressed (Table I). Thus, although an abundant [M ϩ H] ϩpeptide ion at m/z 1717.9 was observed in all MALDI-TOF mass spectra, a less abundant satellite ion at m/z 1703.9 attributed to the gB4 and gB6 gene products (16) was consis-tently seen. In addition, in some spectra, the m/z 2188.1 ion (unique to the gA gene product) was seen in addition to the m/z 2218.1 ion common to the gB1-6 gene products. It seems improbable that the minor polymorphisms among the GARP genes are functionally significant. Possibly, the co-expression of several GARP genes is necessary to achieve a high level of protein expression from regions of low transcriptional activity. In any case, the co-expression of two or more genes encoding major GPI-anchored surface glycoproteins in procyclic T. brucei (7,38,39) and epimastigote T. cruzi (40) has been previously described, and the expression of GARP genes in T. congolense appears to follow this pattern.
The sequence coverage by the MALDI-TOF tryptic mass maps of native GARP was low (27.9%), and only four distinct peptides were identified (Table I). These data suggested that the carbohydrate content of GARP might be protecting the glycoprotein from digestion by trypsin. Consistent with this view, deglycosylation by treatment with mild acid or aq.HF prior to tryptic digestion led to the identification of three and four more peptides and 44.2 and 43.3% sequence coverage, respectively ( Table I).
Analysis of the PI Moieties of GARP GPI Anchors-To analyze the PI component of the GPI anchors, GARP was Western blotted to PVDF membrane and subjected to solid-phase nitrous acid deamination to release the PI components. These components were extracted into butan-1-ol, purified by silica chromatography, and analyzed by negative ion ES-MS (Fig.  4A). Pseudomolecular [M Ϫ H] Ϫ ions at m/z 861, 863, and 865 were observed. ES-MS-MS of these ions produced daughter spectra typical of that shown for m/z 861 in Fig. 4B. In all cases, the spectra showed an abundant m/z 153 [glycerol-2,3-cyclic P] Ϫ ion and only a trace of the m/z 241 [inositol-1,2-cyclic P] Ϫ ion. This is characteristic of inositol-acylated PI structures (7). This type of PI structure was confirmed when GARP was shown to be resistant to the enzyme PI-phospholipase C (data not shown), which cannot cleave the phosphodiester bond of phosphatidylinositol whether or not there is a fatty acid at the 2-position of inositol (41). The major C18:0 carboxylate fragment ion at m/z 283 was present in all spectra as was the m/z 419 [sn-1-stearoylglycerol-2,3-cyclic P] Ϫ ion. These data suggest that all of the PIs contain a stearic acid esterified to the sn-1 position of the glycerol backbone as part of a sn-1-stearoyl-2-L-glycerol unit. Other informative daughter ions include m/z 577 (m/z 579 and 581 for daughter ions of m/z 863 and 865, respectively), which corresponds to the loss of stearate, and m/z 503 (m/z 505 and 507 for daughter ions of m/z 863 and 865, respectively), which corresponds to the loss of sn-1-stearoyl-2-L-glycerol. As previously noted (7), the fatty acid present on the inositol 2-hydroxyl (C18:2, C18:1, and C18:0 for m/z 861, 863, and 865, respectively) does not produce an abundant carboxylate fragment ion in the daughter ion spectra. The proposed structures of the main PI moieties released from GARP are shown in Fig. 4C. A weak ion at m/z 837 was also recorded in Fig. 4A, and this most probably corresponds to a PI species with palmitate (C16:0) attached to the inositol ring.
It is often suggested in the literature that the fatty acid found esterified to the 2-OH of the inositol ring is exclusively palmitate (C16:0). Although this may be the case in higher eukaryotes (41)(42)(43), the few data available from the protozoa suggest that this is not the case. Thus, the GPI anchors of Plasmodium falciparum contain some myristate (C14:0) as well as palmitate (44,45), and the GPI anchors of the T. brucei EP and GPEET procyclins contain C18:0, C18:1, C18:2, and C16:0 fatty acids (in that order) on the inositol ring (7). The data described here for T. congolense GARP are very similar to those for the T. brucei procyclins with the exception that the order of abundance is C18:2 Ͼ C18:1 Ͼ C18:0 Ͼ C16:0. The significance of longer and unsaturated fatty acids in this location is unclear. CONCLUSIONS Based on the data described in this paper, we can propose a partial structure for T. congolense GARP (Fig. 5). The model proposes that GARP has a GPI anchor similar to that of T. brucei procyclin but with a higher degree of unsaturation in the fatty acid attached to the inositol ring. In contrast to procyclin, GARP does not contain a highly modified GPI anchor rich in poly-N-acetyllactosamine repeats. Instead, GARP contains two large Man and Gal-rich oligosaccharides linked via phosphodiester bonds to two Thr residues. Thus, GARP contains some features of procyclin together with features described in the Leishmania proteophosphoglycans (31,32), T. cruzi glycoprotein 72 (33), and Entamoeba histolytica proteophosphoglycans (46), namely Ser/Thr-linked phosphoglycan chains. The fundamental differences between procyclin structure and GARP structure in terms of post-translational modifications calls into question the relevance of the rescue of procyclin-deficient T. brucei procyclic cells with GARP genes (15). Thus, one would expect GARP to be processed similar to procyclin and to bear a highly substituted GPI anchor but no phosphoglycan chains when expressed in T. brucei and to bear little resemblance to GARP expressed in T. congolense. Monoclonal antibodies directed against the surface of T. congolense cells invariably are glycan-specific (12,13), and the large phosphothreonine-linked Man and Gal-rich oligosaccharides probably include the epitopes involved. If so, these or related structures appear to be present on the recently described protease-resistant surface molecule of procyclic T. congolense, which also binds these monoclonal antibodies (14). Judging from monoclonal antibody binding, these carbohydrate epitopes are present on trypanosomes early and late in tsetse infection but are usually absent from those in established mid-gut infections (14). The functional significance of these glycans is presently unclear.