ABH Antigens Research Articles

Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera.

The GII.4 noroviruses (NoVs) are a single genotype that is responsible for over 50% of NoV gastroenteritis epidemics worldwide. However, GII.4 NoVs have been found to undergo antigenic drifts, likely selected by host herd immunity, which raises an issue for vaccine strategies against NoVs. We previously characterized GII.4 NoV antigenic variations and found significant levels of antigenic relatedness among different GII.4 variants. Further characterization of the genetic and antigenic relatedness of recent GII.4 variants (2008b and 2010 cluster) was performed in this study. The amino acid sequences of the receptor binding interfaces were highly conserved among all GII.4 variants from the past two decades. Using serum samples from patients enrolled in a GII.4 virus challenge study, significant cross-reactivity between major GII.4 variants from 1998 to 2012 was observed using enzyme-linked immunosorbent assays and HBGA receptor blocking assays. The overall abilities of GII.4 NoVs to bind to the A/B/H HBGAs were maintained while their binding affinities to individual ABH antigens varied. These results highlight the importance of human HBGAs in NoV evolution and how conserved antigenic types impact vaccine development against GII.4 variants.

No evidence for a direct effect of von Willebrand factor's ABH blood group antigens on von Willebrand factor clearance

One of the major determinants of von Willebrand factor (VWF) plasma levels is ABO blood group status, and individuals with blood groupO have ~25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated. To determine whether clearance of VWF is directly dependent on the presence of ABH antigens on VWF. Three type3 von Willebrand disease (VWD) patients were infused with Haemate-P, and the relative loading of VWF with ABH antigens at different time points was measured. VWF-deficient mice were injected with purified plasma-derived human VWF obtained from donors with either blood groupA, blood groupB, or blood groupO. In mice, we found no difference in clearance rate between plasma-derived blood groupA, blood groupB and blood groupO VWF. Faster clearance of the blood groupO VWF present in Haemate-P infused in type3 VWD patients would have resulted in a relative increase in the loading of VWF with A and B antigens over time. However, we observed a two-fold decrease in the loading with A and B antigens in two out of three patients, and stable loading in the third patient. There is no direct effect of ABH antigens on VWF in VWF clearance. We demonstrate that, in a direct comparison within one individual, blood groupO VWF is not cleared faster than blood groupA or blood groupB VWF. Clearance differences between blood groupO and non-blood groupO individuals may therefore be related to the blood group status of the individual rather than the ABH antigen loading on VWF itself.

Stable Blood Group Chimerism in Recipients of ABO Incompatible Allogeneic Stem Cell Transplantation

Background: The ability to express the ABH antigens in oral mucosa and saliva is present in about 80% of the general population (hence called secretors) and is regulated by the Sec gene on chromosome 19q13.3, which encodes for the enzyme fut2 (α(1,2)Fucosyltransferase), resulting in the expression of blood types in body fluids, including saliva. The gene encoding for the ABH antigens that determines ABO blood type, is located on chromosome 9q34, and transcribes to the enzyme fut1. While fut1 is mainly expressed in erythroid tissue, fut2is expressed in secretory cells. The aim of the present study was to evaluate the ABO type in saliva and red blood cells of patients undergoing allogeneic SCT from ABO mismatched donorsMethods: Secretion status and ABO type in saliva and blood were analyzed in patients with different hematological malignancies undergoing alloSCT from ABO incompatible donors and from healthy donors serving as controls. All study participants signed informed consent. For the determination of ABO type in saliva, following mouth rinse, 5 cc of saliva were collected from each participant into fresh tube and immediately frozen at -800C. Saliva ABH antigens were extracted and enzymes were inactivated. Secretor status and ABO type in patients’ saliva were determined by inhibition test. Agglutination of diluted A, B or O typed donor red cells was tested macroscopically in the presence or absent of the extracted ABH antigens pre-incubated with anti-A, anti-B or anti-H, respectively. ABO blood type was routinely determined in patients at least every 2 weeks and time to ABO type conversion was recorded as along with all transplant-related clinical dataResults: The study cohort included 30 patients (16 males and 14 females; median age 54.2, 18.8-68.5), who underwent alloSCT between Dec 2009 and Feb 2014 from an ABO incompatible donor and were available for routine follow-up. Median follow-up time from transplant to last ABO determination in saliva was 613 days (153-2789).Donors were matched related in 11, matched unrelated in 16 and mismatched unrelated in 3 cases. All grafts were from mobilized peripheral blood. Transplant from major, minor and bidirectional ABO incompatible donors was present in 9, 16 and 5 recipients, respectively. All patients engrafted. Chimerism analysis at day 30 and 100 post transplant by PCR for STR was 100% in 24/25 and 23/25 of tested patients, respectively. Median days to ABO type conversion were 64 (21-290). Of 30 patients, 26 were found to be secretors (87%).In the secretor group, 29/30(96.6%) retained original blood group in the saliva, while one patient originally typed as AB and transplanted from an A type donor, did not retain his original AB blood type in the saliva. It is not clear whether the lack of B-antigen is attributed to the acute mucosal GvHD or to a true conversion of the ABH antigen expression in the salivaConclusion: To the best of our knowledge, this is the first report of stable chimerism of the ABO blood groups post ABO-incompatible allogeneic transplantation, such that the majority of recipients (96.6%) retained the recipient ABO group in the saliva, while expressing the donor ABO group in the blood. The significance of these findings and correlation with long-term outcome need to be further studied in larger patient cohort DisclosuresNo relevant conflicts of interest to declare.

Correlation of ABH blood group antigens secretion with Helicobacter pylori infection in Pakistani patients.

A and B blood group antigens are fucosylated carbohydrate present on human erythrocytes and body secretions. Their presence in body secretions depends on the expression of a dominant allele of secretor gene FUT2 and is correlated with susceptibility to various infectious and non-infectious diseases. We investigated the correlation of blood group and ABH antigen secretion with Helicobacter pylori infection and gastroduodenal symptoms and analysed the distribution of babA gene among ABH secretors and non-secretors. Two hundred and ninety patients who underwent gastroduodenal endoscopy during 2011 to 2012 participated. Gastric biopsy, saliva and blood samples were obtained from every patient. Gastric biopsies were subjected to rapid urease test and PCR for the detection of H.pylori and babA gene. Blood grouping and ABH antigens secretions were determined by Lewis blood group phenotyping and haemagglutination inhibition test. 50.34% of patients were ABH antigen secretors and 45.51% non-secretors. Distribution analysis of blood group revealed that 40 blood group B, 67 blood group A 20 blood group O and 19 blood group AB patients secreted ABH antigens in saliva. Fifty-six blood group O, 19 blood group B, 32 blood group A and 17 blood group AB patients were non-secretors. Gastroduodenal complaints were common among non-secretors. Sixty-two percent of patients with a combination of duodenal ulcer and gastro-oesophageal reflux and 54% of patients with gastritis were non-secretors. Of 290 samples, 31.02% were positive for H.pylori. Thirty percent of these tested positive for babA gene; the majority belonged to non-secretor blood group O. Our results suggest that the infection of H.pylori is correlated with ABO blood groups and blood group antigens secretion in body fluids.

Importance of including O cells in reverse grouping in detection of Bombay phenotype (Oh)

Background: Bombay blood group is the most frequently asked rare blood in India. It is characterised by absence of normal ABH antigens and have corresponding antibodies in serum. This blood group is suspected when reagent O cells show agglutination in reverse or back typing or during antibody screening. We present a case of Bombay phenotype which was initially mistyped as O group. Methods: Patient was having Iron deficiency anaemia and presented to our centre with signs and symptoms of anaemia. Two units of packed red cells were requested by physician. On routine typing strong reaction with O cells in reverse grouping and pan-reactive picture on antibody screen, which led to the suspicion of Oh phenotype. Results/conclusions: We report a case of Bombay blood group referred to our centre which was managed successfully by contacting voluntary donor though rare donor registry. We highlight the importance of following standard protocol for proper pretransfusion testing including blood grouping and also close coordination among blood banks across the country.“Bombay phenotype\

Faecal microbiota composition in adults is associated with the FUT2 gene determining the secretor status.

The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota.

Molecular Deciphering of the ABO System as a Basis for Novel Diagnostics and Therapeutics in ABO Incompatible Transplantation

In recent years ABO incompatible kidney transplantation (KTx) has become a more or less clinical routine procedure with graft and patient survival similar to those of ABO compatible transplants. Antigen-specific immunoadsorption (IA) for anti-A and anti-B antibody removal constitutes in many centers an important part of the treatment protocol. ABO antibody titration by hemagglutination is guiding the treatment; both if the recipient can be transplanted as well as in cases of suspected rejections if antibody removal should be performed. Despite the overall success of ABO incompatible KTx, there is still room for improvements and an extension of the technology to include other solid organs. Based on an increased understanding of the structural complexity and tissue distribution of ABH antigens and the fine epitope specificity of the ABO antibody repertoire, improved IA matrices and ABO antibody diagnostics should be developed. Furthermore, understanding the molecular mechanisms behind accommodation of ABO incompatible renal allografts could make it possible to induce long-term allograft acceptance also in human leukocyte antigen (HLA) sensitized recipients and, perhaps, also make clinical xenotransplantation possible.

English

INTRODUCTION: The blood group serology plays an important role in transfusion medicine. The discovery of a rare blood group, Bombay (Oh) Phenotype by Bhende et al in 1952 in Mumbai formerly (Bombay) was important in the field of immunohematology. The discovery later helped Watkins and Morgan1 (1959) and Gerard et al2 (1982) to elucidate biosynthetic pathway for ABH and Lewis (Le) antigens suggesting the secretor (Se) and (H) are closely linked structural genes. Recently, molecular genetic studies were carried out to determine the role of the H, Se and Le genes in the expression of H antigen in secretions and Lewis blood group antigen on erythrocytes. 3,4. It is known that the precursor protein from which all blood groups are formed is termed as the “H” Antigen. This ‘H’ antigen either translates into ‘A’ antigen (blood group A) or into ‘B’ Antigen (blood group B) or into both A and B Antigens (blood group AB) or it remains as ‘H’(blood group ’O’).In the case of Bombay blood group, there is absence of ‘H’ Antigen itself. Therefore ‘A’, ‘B’, ‘AB’ and ‘O’ which are all different manifestations of ‘H’ are alien to persons with Bombay blood group. The Bombay blood group is termed ‘Oh’ meaning absence of ‘H’ Antigen. CASE REPORT: A 25 year old Post C.S. 2nd gravidae, at term pregnancy was referred from Purulia District Hospital and admitted in our hospital through emergency. The patient gave history of irregular antenatal checkup and did not consume iron folate tablets. Her menstrual history was normal. Her previous pregnancy and caesarean section were uneventful with no previous history of blood transfusion. There was no family history of Thalassemia or blood transfusion. On examination the patient was severely anaemic (Hb-4.5 gm %) with tachycardia and pedal oedema of lower limbs. Her blood pressure was 110/68 mm Hg Obstetric examination revealed full term post caesarean pregnancy with longitudinal lie, cephalic presentation, F.H.S.-146/min, regular with mild uterine contraction. Per vaginal examination suggested that she was in early labour. Immediate decision for blood transfusion and termination of pregnancy by LUCS was taken. The blood was sent for grouping and cross matching to our Blood Bank where she was diagnosed to have the Bombay Blood group after reverse grouping. As this group is very rare, we could not find a single unit of this group in Bankura district and immediate blood transfusion could not be performed. Ultimately, after 4hours of admission, LUCS was performed without blood. After emergency caesarean section the first 72 hours were uneventful. On the 4 th post-operative day, the

Failure of Neonatal B-Cell Tolerance Induction by ABO-Incompatible Kidney Grafts in Piglets

ABO-incompatible (ABOi) infant heart transplantation results in B-cell tolerance to graft A/B antigens, confirming human susceptibility to acquired immunologic or "neonatal" tolerance as described originally in murine models. Starting with this clinical observation, we sought to model neonatal ABOi organ transplantation to allow mechanistic studies of tolerance. Plasma anti-A/B antibodies were measured over time in piglets to establish developmental antibody kinetics. Blood group O piglets received kidney allografts from group A (AO-incompatible) or group O (AO-compatible) donors under cyclosporine immunosuppression. Anti-A antibodies were measured serially after transplantation; A/H antigen expression and allograft rejection were assessed in graft biopsies. Anti-A antibodies developed in naïve piglets in a kinetic pattern analogous to human infants; anti-B remained low. After transplantation, anti-A antibodies developed similarly in AO-incompatible and AO-compatible groups and were not suppressed by cyclosporine. A/H antigen expression was persistent in all graft biopsies; however, A/H antigens were not detected in vascular endothelium. Cellular and antibody-mediated rejection was absent or minimal in early and late biopsies in both groups, with one exception. Naturally delayed isohemagglutinin production in piglets is analogous to the developmental kinetics in human infants. However, in contrast to deficient anti-A antibody production as seen long-term after "A-into-O" infant heart transplant recipients, normal anti-A antibody production after "A-into-O" piglet kidney transplantation indicates that tolerance did not develop despite graft A antigen persistence. These findings suggest that the impact on the host immune system of exposure to nonself ABH antigens during early life in human heart versus porcine kidney grafts may depend on expression in vascular endothelium.

Noroviruses and histo‐blood groups: the impact of common host genetic polymorphisms on virus transmission and evolution

Noroviruses (NoVs) are recognized as a leading cause of human gastroenteritis worldwide. Infection occurs following the ingestion of contaminated food or, most often, through direct contact from person to person. However, not all individuals are equally sensitive to these viruses. Indeed, NoVs use glycans of the ABH and Lewis histo-blood group antigen family (HBGAs) as attachment factors. At the epithelial level, the synthesis of these HBGAs requires the action of several glycosyltransferases that are encoded by the ABO, FUT2, and FUT3 genes. The combined polymorphism at these three loci dictates sensitivity to NoV infection because the attachment profile to these glycans varies among strains. Structural analysis of the capsid protein interaction with HBGAs reveals distinct modes of binding for strains of genogroups I and II but high conservation within each genogroup, whereas minor amino acid changes are sufficient to generate modifications of HBGA-binding specificities or affinities. Such modifications therefore induce changes in the spectrum of susceptible individuals. Studies of NoV-HBGA interactions together with phylogenetic analyses and the epidemiologic survey of strains indicate that NoV transmission and evolution depend on both the establishment of herd immunity and the genetic resistance of many individuals, which confers herd innate protection by restricting NoV circulation.

Blood Type B antigen modulates cell migration through regulating cdc42 expression and activity in HaCaT cells

ABO blood group is determined by carbohydrate antigens, called ABH antigens. It has been known that the change of carbohydrate antigen expression, including ABH antigens, has correlation with the tumor metastasis and survival; however, the exact mechanism remains to be elucidated. ABH antigens are expressed not only in blood cells but also in several tissues. In epidermis, ABH antigen is expressed in the uppermost spinous and granular layer. We investigated the role of ABH antigens on the cell migration of HaCaT keratinocytes, which express B antigen. Knock-down of B antigen expression by small interference RNA of FUT1 inhibited HaCaT cell migration. At that time, we found that lamellipodia and actin fiber were also reduced by knock-down of B antigen expression. The transcription of cdc42, a kind of Rho GTPase which plays a key role in actin polymerization, was reduced by down-regulated B antigen expression. Furthermore, the reduced B antigen expression also inhibited the interaction of cdc42 and N-WASP. Collectively, our data provide a clue how ABH antigens regulate the cell migration mechanism.

Expression of the FUT2 gene and CD44 marker in patients with oral lesions

The aim of this work was to investigate the FUT 2 gene, the secretor status and the expression of CD44 protein in epithelial cells obtain from saliva samples from patients with oral lesions (benign, pre-cancerous and cancerous lesions, n=94). We analyzed polymorphisms of the FUT2 gene by allele specific oligonucleotide–polymerase chain reaction. The FUT2 gene encodes the α(1,2) fucosyltransferase (Se enzyme) that regulates the expression of ABH antigens in secretions. Generally speaking, being a non-secretor has several disadvantages with regard to metabolism and immune function. In this study, we found that oral pre-cancerous and cancerous lesions were increased among individuals with non-secretor status and nonsense mutation 428G→A. Fifty-one percent of the patients with oral pre-malignant and malignant lesions were non-secretors, in contrast with the healthy population (OR=3.44). We observed a marginal association between secretor status and these lesions. Our study suggests that the lack of wild type FUT2 gene and a non-secretor status appear to be an associated risk marker for the development of oral cancer in patients with oral lesions.One of the genes involved in tumour processes is CD44, which appears to be one of the most promising candidates as a cancer diagnosis marker. We investigated, using confocal microscopy, the expression of CD44 protein in epithelial cells obtained from saliva samples from patients with oral lesions. The results obtained showed fluorescence corresponding to the presence of CD44 protein in samples from patients diagnosed with cancer and pre-cancer. These findings indicate that overexpression of CD44 molecule analyzed could be considered as a marker of risk in individuals with oral lesions.

The summary of FUT1 and FUT2 genotyping analysis in Chinese para‐Bombay individuals including additional nine probands from Guangzhou in China

The para-Bombay phenotype is characterized by the absence or weak expression of ABH antigens on the surface of red blood cells, but normal expression in saliva. The para-Bombay phenotype of the nine Chinese probands was identified by standard serologic techniques. The coding regions of FUT1 and FUT2 genes were amplified by polymerase chain reaction and then directly sequenced. ABO genotyping was performed by polymerase chain reaction with sequence-specific priming method. The FUT1 and FUT2 genotypes and the distribution in all reported Chinese para-Bombay individuals including our study were also summarized. Five FUT1 genotypes, h1h3 (n = 3), h1h2 (n = 3), h1h1 (n = 1), h3h3 (n = 1), and h2h3 (n = 1), and three functional FUT2 genotypes, Se(357) Se(357) (n = 4), Se(357) Se(357, 716) (n = 4), and Se(357) Se(357, 385) (n = 1) described before were identified in nine probands. The review of the literature shows that a total of 17 FUT1 alleles and four FUT2 alleles (Se(357), Se(357,716), Se(357 385), Se) have been identified in Chinese para-Bombay individuals. The four FUT1 alleles, h1 (547delAG), h2 (880delTT), h3 (C658T), and h4 (C35T; A980C) are most prevalent, which account for more than 90% of all allele counts and are essential to be involved when developing para-Bombay genotyping kit for Chinese.

Forensic validation of the modified ABO genotyping method using a DNA chip

ABO typing is effective in several forensic investigations, including the identification of unknown cadavers. When the serological method cannot be used because of the decomposition of ABH antigens, ABO genotyping of DNA is often performed. Previously, we reported a novel ABO genotyping method using a DNA chip as a proof of concept. This chip can simultaneously detect single nucleotide polymorphisms in the ABO gene and a part of the primate-specific D17Z1 sequence for human identification. In the present study, this method was modified and further validated for forensic use. We demonstrated that the modified method can correctly perform ABO genotyping and human identification if the appropriate amount of template (>0.5ng of DNA) is analyzed. Moreover, it was found that this chip method can be used to type highly degraded DNA. This method is expected to be a useful supplemental tool for the identification of individuals from highly decomposed samples.

Relationship betweenABOgenotype andAantigen expression on platelets

Although platelets (PLTs) are known to express ABH antigens, the extent of expression is different on PLTs compared with red blood cells and the relationship between PLT ABH expression and genotype has not been thoroughly investigated. We measured blood group H and A antigens on PLTs from 100 normal volunteers using fluorescent-conjugated reagents and flow cytometry. Individuals were also genotyped at the ABO locus using a commercially available genotyping system. Expression of A and H antigen varied widely on PLTs from different individuals. Among group A and AB persons, H antigen expression was significantly greater than A antigen (p < 0.0001). The ratio of H-to-A antigen expression varied more than 100-fold in a predictable fashion according to genotype with values lowest in A1/A1 < A1/O < A2/A2 < A2/O. H and A antigen expression was unaffected by secretor status. The proportion of PLTs with high A expression also varied directly according to genotype. Blood group A and H antigen expression on PLTs varies in a predictable fashion according to genotype. Flow cytometry and genotyping identify individuals who strongly express A antigens, a finding that may be relevant to clinical PLT transfusion across ABO barriers.

Anti-H can trigger apoptosis and down-regulate FUT1 expression in erythroid differentiated K562 cells without complement mediation

The reason why delayed RBC engraftment and pure red cell aplasia (PRCA) develop only in some but not all recipients of major ABO-incompatible hematopoietic stem cell transplantation (HSCT) remains elusive and the underlying mechanisms are not fully understood. Understanding how incompatible erythroid blood group antibodies (Abs) interact with ABH antigens (Ags) of grafts, and investigating how to induce artificially accommodation of grafts are of obvious importance in transplantation immunology.The effects of anti-H on proliferation, apoptosis, and α-(1,2)-fucosyltransferase gene (FUT1) expression in erythroid differentiated K562 cells were analyzed by the MTT assay, Annexin V/PI staining, and quantitative RT-PCR method.The growth of erythroid differentiated K562 cells was significantly suppressed when anti-H dilution was ≤1:8 (P<0.001, as compared with 1:16). Under the complement-free culture conditions, the apoptotic ratio of erythroid differentiated K562 cells was significantly increased when anti-H dilution was ≤1:16 (P<0.05, as compared with 1:32). The apoptosis was not only closely associated with anti-H dilution (F=138.991, P<0.001), but also correlated with treated time (F=583.249, P<0.001), which indicated typical dose- and time-dependent effects. Under the complement-free culture conditions, the FUT1 mRNA expression level was also suppressed when anti-H dilution was ≤1:16 (P<0.05, as compared with 1:32), which also manifested in typical dose-dependent (F=130.356, P<0.001) and time-dependent (F=1432.00, P<0.001) effects.The results confirm that anti-H can trigger apoptosis and down-regulate FUT1 expression in erythroid differentiated K562 cells without complement mediation. The findings suggest that anti-H could accommodate grafts through triggering apoptosis and down-regulating Fut1 expression to reduce ABH antigens.

Mapping the N-glycome of human von Willebrand factor

vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn(2635) site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.

Expression of the carcinoma markers: the sialylated Lewis A and X carbohydrate antigens in normal laryngeal surface epithelium and submucosal glands from old humans

Aberrant surface expression of the carbohydrate ABH and Lewis antigens are often used as markers for the diagnosis of cancer, but while the distribution of these histo-blood group antigens is relatively well-described in tissues and organs from young and middle-aged humans little is known of their expression in old age. The objective for this study was to estimate if the Lewis A and X antigens together with their sialylated modifications, are expressed in sections of normal laryngeal tissue from old humans. Antibodies directed against the tumor markers Sialyl Lewis A and Sialyl Lewis X showed positive reaction in the surface epithelia from normal larynx autopsies obtained from people aged 77-90 years. The sialylated and non-sialylated Lewis A antigens were more frequently expressed in the pseudostratified epithelium than in squamous surface epithelium. Both the sialylated and the non-sialylated carbohydrates were stained in the submucosal glands in all the autopsies. In conclusion, visualization of Lewis tumor markers in the larynx should be interpreted with great care, as they may be present in normal laryngeal epithelial cells from old humans.

Structural Basis for the Recognition of Lewis Antigens by Genogroup I Norovirus

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le(a)) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le(a), Le(b), H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le(a) antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le(a) antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le(a) antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le(a) antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galβ1-3GlcNAc or Galβ1-3GalNAc as basic motifs was commonly better than that to canonical Galβ1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galβ1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.