FORSCells: 40-days fixed prepared reagent for detection of anti-Forssman in humans.

In 2012, the FORS system was accepted by the International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is most commonly found on sheep red blood cells (RBC) but rare in human RBC. Anti-Fs antibodies (Ab) are naturally occurring in human sera and are predominantly IgM but they can also be IgG. To this day, the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1-positive RBC available to use for anti-Fs screening in large populations. This study was designed to produce FORS1-positive cells viable for 40 days use in the anti-Fs screening. Three to 5% FORS1-positive cells were produced using sheep's blood and CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated in more than three independent experiments of ABO titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. To demonstrate that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification were carried out in a patient and donor population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good FORS1 Ag preservation was established, and minimal haemolysis was observed. In conclusion, a novel and easy-to-produce reagent has been developed and submitted to a patent with stable FORS1 Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Ab in large populations.

Blood group ABO gene-encoded A transferase catalyzes the biosynthesis of FORS1 antigen of FORS system upon Met69Thr/Ser substitution.

Blood group A/B glycosyltransferases (AT/BTs) and Forssman glycolipid synthase (FS) are encoded by the evolutionarily related ABO (A/B alleles) and GBGT1 genes, respectively. AT/BT and FS catalyze the biosynthesis of A/B and Forssman (FORS1) oligosaccharide antigens that are responsible for the distinct blood group systems of ABO and FORS. Using genetic engineering, DNA transfection, and immunocytochemistry and immunocytometry, we have previously shown that the eukaryotic expression construct encoding human AT, whose LeuGlyGly tripeptide at codons 266 to 268 was replaced with FS-specific GlyGlyAla tripeptide, induced weak appearance of FORS1 antigen. Recently, we have shown that the human AT complementary DNA constructs deleting exons 3 or 4, but not exons 2 or 5, induced moderate expression of FORS1 antigen. The constructs containing both the GlyGlyAla substitution and the exon 3 or 4 deletion exhibited an increased FS activity. Here, we report another molecular mechanism in which an amino acid substitution at codon 69 from methionine to threonine or serine (Met69Thr/Ser) also modified enzymatic specificity and permitted FORS1 biosynthesis. Considering that codon 69 is the first amino acid of exon 5 and that the cointroduction of Met69Thr and GlyGlyAla substitutions also enhanced FS activity, the methionine substitutions may affect enzyme structure in a mode similar to the exon 3 or 4 deletion but distinct from the GlyGlyAla substitution.

Prevalence of antibodies to a new histo-blood system: the FORS system.

BACKGROUND: In 1987, three unrelated English families were reported with a putative blood subgroup called Apae. Swedish researchers later found evidence leading to abolishment of the Apae subgroup and establishment instead of the FORS blood group system (System 31 - ISBT, 2012). It is important to know the prevalence of antibodies in order to make the best decisions in transfusion medicine. Cells expressing the Forssman saccharide, such as sheep erythrocytes, are needed to detect the anti-Forssman antibody. The aim of this study was to define the prevalence of human anti-Forssman antibody. MATERIALS AND METHODS: Plasma samples from 800 individuals were studied. Sheep erythrocytes or Forssman "kodecytes" were mixed with the plasma samples using the tube technique. Plasma from an Apae individual was used as a negative control and monoclonal anti-Forssman antibody (M1/22.25.8HL cell line supernatant) was used as the positive control. RESULTS: Of the 800 individuals tested, one was negative for the presence of anti-Forssman antibody. We compared the anti-Forssman antibody reaction pattern between genders and found that males have weaker reactions than females, both at room temperature (p=0.026) and at 37 °C (p=0.043). We also investigated the reaction pattern of anti-Forssman antibody in relation to ABO and Rh blood group types without finding any significant differences. DISCUSSION: Sheep erythrocytes are suitable for searching for human anti-Forssman antibody. The quantity of anti-Forssman antibodies in plasma is higher in females than in males. In the population (n=800) studied here, we found one individual lacking the anti-Forssman antibody. These results contribute to the data already published, confirming that FORS is a rare blood group.

Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases.

A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars.

Monoclonal anti-A activity against the FORS1 (Forssman) antigen.

BACKGROUND: The FORS blood group system (originally recognized as the Apae phenotype) was discovered by sporadic activity against polyclonal anti-A reagents and activity against the lectin Helix pomatia. The extent of monoclonal anti-A reagent activity against the FORS1 antigen is serologically and immunochemically incomplete. STUDY DESIGN AND METHODS: In the absence of natural FORS1-positive red blood cells (RBCs), kodecytes were created with synthetic disaccharide and pentasaccharide Forssman function-spacer-lipid (FSL) constructs, Fsdi -kodecytes, and FORS1-kodecytes, respectively. FSL constructs were also applied to solid surfaces and used in solid-phase enzyme immunoassays. A range of characterized monoclonal anti-A and anti-B reagents were then serologically and immunochemically characterized against these Forssman antigens. Polyclonal human anti-A, anti-B, the lectin H. pomatia serologic reagents; and canine RBCs were used as serologic controls. RESULTS: None of 19 different monoclonal anti-A reagents were able to detect the pentasaccharide Forssman on FORS1-kodecytes, while three reagents were able to detect disaccharide Forssman on Fsdi -kodecytes. Most anti-A reagents were immunochemically reactive with both the di- and the pentasaccharide Forssman antigens in the solid-phase assays. Historic polyclonal human anti-A and the lectin H. pomatia reacted strongly with the FORS1-kodecytes, correlating with the discovery of the Apae phenotype and supporting the use of FORS1-kodecytes as FORS1 surrogates. CONCLUSIONS: Monoclonal anti-A reagents, despite showing reactivity against the FORS1 antigen in solid-phase assays are unlikely to cause the agglutination of FORS1 antigen-positive RBCs.

Synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives by a one-pot glycosylation procedure.

The synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives thereof by using a one-pot glycosylation and polymer-assisted deprotection is described. The Forssman antigen pentasaccharide, composed of GalNAcα(1,3)GalNAcß(1,3)Galα(1,4)Galß(1,4)Glc, was recently identified as a ligand of the lectin SLL-2 isolated from an octocoral Sinularia lochmodes. The chemo- and α-selective glycosylation of a thiogalactoside with a hemiacetal donor by using a mixture of Tf(2)O, TTBP and Ph(2)SO, followed by activation of the remaining thioglycoside, provided the trisaccharide at the reducing end in a one-pot procedure. The pentasaccharide was prepared by the α-selective glycosylation of the N-Troc-protected (Troc=2,2,2-trichloroethoxycarbonyl) thioglycoside with a 2-azide-1-hydroxyl glycosyl donor, followed by glycosidation of the resulting disaccharide at the C3 hydroxyl group of the trisaccharide acceptor in a one-pot process. We next applied the one-pot glycosylation method to the synthesis of pentasaccharides in which the galactosamine units were partially and fully replaced by galactose units. Among the three possible pentasaccharides, Galα(1,3)GalNAc and Galα(1,3)Gal derivatives were successfully prepared by the established method. An assay of the binding of the synthetic oligosaccharides to a fluorescent-labeled SLL-2 revealed that the NHAc substituents and the length of the oligosaccharide chain were both important for the binding of the oligosaccharide to SLL-2. The inhibition effect of the oligosaccharide relative to the morphological changes of Symbiodinium by SLL-2, was comparable to their binding affinity to SLL-2. In addition, we fortuitously found that the synthetic Forssman antigen pentasaccharide directly promotes a morphological change in Symbiodinium. These results strongly indicate that the Forssman antigen also functions as a chemical mediator of Symbiodinium.

Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system.

In analogy with histo-blood group A antigen, Forssman (Fs) antigen terminates with α3-N-acetylgalactosamine and can be used by pathogens as a host receptor in many mammals. However, primates including humans lack Fs synthase activity and have naturally occurring Fs antibodies in plasma. We investigated individuals with the enigmatic ABO subgroup A(pae) and found them to be homozygous for common O alleles. Their erythrocytes had no A antigens but instead expressed Fs glycolipids. The unexpected Fs antigen was confirmed in structural, serologic, and flow-cytometric studies. The Fs synthase gene, GBGT1, in A(pae) individuals encoded an arginine to glutamine change at residue 296. Gln296 is present in lower mammals, whereas Arg296 was found in 6 other primates, > 250 blood donors and A(pae) family relatives without the A(pae) phenotype. Transfection experiments and molecular modeling showed that Agr296Gln reactivates the human Fs synthase. Uropathogenic E coli containing prsG-adhesin-encoding plasmids agglutinated A(pae) but not group O cells, suggesting biologic implications. Predictive tests for intravascular hemolysis with crossmatch-incompatible sera indicated complement-mediated destruction of Fs-positive erythrocytes. Taken together, we provide the first conclusive description of Fs expression in normal human hematopoietic tissue and the basis of a new histo-blood group system in man, FORS.

Synthesis of a Forssman antigen derivative for use in a conjugate vaccine.

The total chemical synthesis of a Forssman antigen analog is described. The pentasaccharide contains a functionalized tether which should facilitate future conjugation with immunogenic proteins. We found that the total synthesis can be efficiently achieved by following a convergent 2+3 strategy, and using N-Troc protected GalNAc thioglycoside as a donor.

Investigation of potential carbohydrate antigen targets for human and baboon antibodies.

BACKGROUND: The continued presence of a primate antibody-mediated response to cells and organs from alpha1,3-galactosyltransferase gene-knockout (GTKO) pigs indicates that there may be antigens other than Gal alpha 1,3Gal (alpha Gal) against which primates have xenoreactive antibodies. Human and baboon sera were tested for reactivity against a panel of saccharides that might be potential antigen targets for natural anti-non-alpha Gal antibodies. METHODS: Human sera (n = 16) and baboon sera (n = 15) of all ABO blood types were tested using an enzyme-linked immunoadsorbent assay for binding of IgM and IgG to a panel of synthetic polyacrylamide-linked saccharides (n = 15). Human sera were also tested after adsorption on alpha Gal immunoaffinity beads. Sera from healthy wild-type (WT, n = 6) and GTKO (n = 6) pigs and from baboons (n = 4) sensitized to GTKO pig organ or artery transplants (of blood type O) were also tested. Forssman antigen expression on baboon and pig tissues was investigated by immunohistochemistry. RESULTS: Both human and baboon sera showed high IgM and IgG binding to alpha Gal saccharides, alpha-lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N-glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on alpha Gal oligosaccharides, there was a reduction in binding to alpha Gal and alpha-lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to alpha Gal saccharides. Baboon sera sensitized to GTKO pigs showed no significant increased binding to any specific saccharide. Staining for Forssman was negative on baboon and pig tissues. CONCLUSIONS: We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti-non-alpha Gal antibodies. The high level of anti-Forssman antibodies in humans, baboons, and pigs, and the absence of Forssman expression on pig tissues, suggest that the Forssman antigen does not play a role in the primate immune response to pigs.

Glycan-binding profile and cell adhesion activity of American bullfrog (Rana catesbeiana) oocyte galectin-1.

The glycan-binding profile of a beta-galactoside-binding 15 kDa lectin (Galectin-1) purified from the oocytes of the American bullfrog, Rana catesbeiana, was studied using 61 pyridyl-aminated oligosaccharides by frontal affinity chromatography. Human blood type-A-hexasaccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta;1-4GlcNAcbeta1-4Galbeta1-4Glc) was found to exhibit the strongest ligand binding to the galectin while Forssman antigen (GalNAcalpha1-3GalNAcbeta1-3Galalpha1-4Galbeta1-4Glc) and type-A-tetrasaccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-4Glc) were also extensively recognized. The kinetics of affinity of galectin-1 to type-A oligosaccharide was analysed by surface plasmon resonance using neoglycoprotein with type-A oligosaccharides. R. catesbeiana oocyte galectin adhered to human rhabdomyosarcoma cells dose dependently and the activity was specifically cancelled by the neoglycoprotein. It was concluded that galectin-1 from R. catesbeiana oocytes possesses different and rare glycan-binding properties from typical members in galectin family.

Complete chemoenzymatic synthesis of the Forssman antigen using novel glycosyltransferases identified in Campylobacter jejuni and Pasteurella multocida.

We have identified an alpha1,4-galactosyltransferase (CgtD) and a beta1,3-N-acetylgalactosaminyltransferase (CgtE) in the lipooligosaccharide (LOS) locus of Campylobacter jejuni LIO87. Strains that carry these genes may have the capability of synthesizing mimics of the P blood group antigens of the globoseries glycolipids. We have also identified an alpha1,3-N-acetylgalactosaminyltransferase (Pm1138) from Pasteurella multocida Pm70, which is involved in the synthesis of an LOS-bound Forssman antigen mimic and represents the only known bacterial glycosyltransferase with this specificity. The genes encoding the three enzymes were cloned and expressed in Escherichia coli as soluble recombinant proteins that can be used to chemoenzymatically synthesize the Forssman antigen, and its biosynthetic precursors, in high yields.

Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin.

Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research.

The relation of serum anti-(GalNAc beta) and -para-Forssman disaccharide IgG levels to the progression and histological grading of gastrointestinal cancer.

UNLABELLED: Earlier we found two unusual IgG-antibody specificities to GalNAc beta and GalNAc beta1-3GalNAc beta (para-Forssman disaccharide, PFdi) carbohydrate ligands in human serum. The aim of the study was to evaluate whether elevated antibody levels are related to the progression of gastrointestinal cancer and the histopathological grading. METHODS: Specific IgG levels were tested in 159 patients with gastric cancer, 88 patients with colorectal cancer and 96 blood donors by the ELISA using synthetic polyacrylaamide (PAA) conjugates, GalNAc beta-PAA and PFdi-PAA. Biochemical and haematological analyses were performed using automatic equipment. RESULTS: The anti-PFdi IgG levels were significantly higher in patients with gastric and colorectal cancer than in donors: in stages II-IV, P = 0.0002 - 0.04 (U-test). The elevated anti-PFdi IgG level was associated with the advanced gastric cancer: in stages II, III, IV vs stage I (P = 0.004 - 0.06) and in case of the tumor size T2 + T3 vs T1 (stages I, II; P = 0.03). Differences in anti-GalNAc beta IgG level were insignificant. No relation between antibody levels and the regional and distant metastases of gastric or colorectal cancer was found. The lower anti-GalNAc beta IgG level was associated with lower-differentiated carcinomas (P = 0.01 - 0.04). Prolonged postoperative changes in the levels of both antibodies during the follow-up were established. An elevation of both antibody levels in patients with gastrointestinal cancer was revealed after a surgical removal of G3-tumors (P = 0.003 - 0.01). The anti-PFdi IgG levels correlated with the levels of the C-reactive protein: r = 0.50, P = 0.003. The anti-GalNAc beta IgG levels correlated with the percentage of peripheral blood monocytes: r = 0.42, P = 0.002. CONCLUSION: The association of the anti-PFdi IgG level with cancer progression suggests the implication of antibodies in the pathogenesis of gastrointestinal cancer. Further studies are required to identify natural targets of antibodies, their relation to other diseases, prognostic significance in cancer.

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model.

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity.

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.

The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency.

5T4 oncotrophoblast antigen is a transmembrane glycoprotein expressed by trophoblast and many carcinomas but not most normal adult tissues. Results from overexpression of human and mouse 5T4 cDNA in cell lines are consistent with it having an influence on adhesion, shape and motility. We show that murine embryonic stem cell lines are 5T4 negative but that there is rapid up regulation of protein and transcripts upon differentiation, including derivatives of each primary germ layer, as evidenced by cell surface FACS, western and RT-PCR analyses. The kinetics of differentiation and 5T4 expression are closely correlated, with early events linking 5T4 expression to changes in motility and morphology. Comparison of 5T4 expression with other ES cell transcript (Oct 3/4; Rex-1) and antigen markers (Forsmann, SSEA-1) establishes 5T4 as a useful marker for the non-destructive detection of early differentiation of ES cells. For example, 'undifferentiated' ES phenotype defined as SSEA-1 positive and 5T4 negative is seven times more efficient at chimera formation than SSEA-1-positive/5T4-positive cells. Thus, 5T4 glycoprotein expression is associated with early differentiative events of ES cells involving altered motility, and it has useful practical consequences for assessing ES potency and studying similar processes in development and metastasis.

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Quantitative studies of the binding of the class II PapG adhesin from uropathogenic Escherichia coli to oligosaccharides.

Binding of the class II PapG adhesin, found at the tip of filamentous pili on Escherichia coli, to the carbohydrate moiety of globoseries glycolipids in the human kidney is a key step in development of pyelonephritis, a severe form of urinary tract infection. An assay based on surface plasmon resonance for quantification of the binding of the class II PapG adhesin to oligosaccharides has been developed. Using this assay dissociation constants ranging from 80 to 540 microM were determined for binding of the PapG adhesin to di-pentasaccharide fragments from the globoseries of glycolipids. A series of galabiose derivatives, modified at the anomeric position, O-2' or O-3', was also investigated. The anomeric position appeared to be the most promising for development of improved inhibitors of PapG-mediated adhesion of E. coli. p-Methoxyphenyl galabioside was found to be most potent (K(d)=140 microM), and binds to PapG almost as well as the Forssman pentasaccharide.

Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A, blood group B, Forssman, and iGb3 antigens and their interaction with substrates.

A terminal alpha1-3 linked Gal or GalNAc sugar residue is the common structure found in several oligosaccharide antigens, such as blood groups A and B, the xeno-antigen, the Forssman antigen, and the isogloboside 3 (iGb3) glycolipid. The enzymes involved in the addition of this residue display strong amino acid sequence similarities, suggesting a common fold. From a recently solved crystal structure of the bovine alpha3-galactosyltransferase complexed with UDP, homology modeling methods were used to build the four other enzymes of this family in their locked conformation. Nucleotide-sugars, the Mn2+ ion, and oligosaccharide acceptors were docked in the models. Nine different amino acid regions are involved in the substrate binding sites. After geometry optimization of the complexes and analysis of the predicted structures, the basis of the specificities can be rationalized. In the nucleotide-sugar binding site, the specificity between Gal or GalNAc transferase activity is due to the relative size of two clue amino acids. In the acceptor site, the presence of up to three tryptophan residues define the complexity of the oligosaccharide that can be specifically recognized. The modeling study helps in rationalizing the crystallographic data obtained in this family and provides insights on the basis of substrate and donor recognition.