Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis of type III and IV structures and NMR characterization of type I-VI antigens

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to VI antigens, which were carried out to probe differences in overall conformation of the molecules. These NMR investigations showed very little difference in the ¹H chemical shifts, as well as ¹H–¹H coupling constants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in solution.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.     

Cloning and characterization of the alpha(1,3/4) fucosyltransferase of Helicobacter pylori

The gastric pathogen Helicobacter pylori can express the histo blood group antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y, whereas a small population express the type I carbohydrates, Lewis A and B. The expression of Lewis A and Lewis X, as in the case of H. pylori strain UA948, requires the addition of fucose in alpha1,4 and alpha1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been identified in each of the genomes sequenced. The characteristic adenosine and cytosine tracts in the amino terminus and repeated regions in the carboxyl terminus are present in the DNA encoding the two UA948fucT genes, but these genes also contain differences when compared with previously identified H. pylori fucTs. The UA948fucTa gene encodes an approximately 52-kDa protein containing 475 amino acids, whereas UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type II carbohydrate acceptors, by UA948FucTa, does not appear to be affected by fucosylation at other sites on the carbohydrate acceptor, but the rate of fucose transfer is affected by terminal fucosylation of type I acceptors. Through mutational analysis we demonstrate that only FucTa is active in this H. pylori isolate and that inactivation of this enzyme eliminates expression of all Lewis antigens.     

Expression of H type 1 antigen of ABO histo-blood group in normal colon and aberrant expressions of H type 2 and H type 3/4 antigens in colon cancer

We have immunohistochemically examined the distribution of the H antigens of type 1, type 2 and type 3/4 chains of the ABO(H) histo-blood group system in human normal colon and in colon cancer using three monoclonal antibodies specific for each of the H type 1/2, H type 2, and the H type 3/4 chain. We unexpectedly found that mucosa of the normal colon from secretors but not that from nonsecretors expressed only H type 1 and did not express H type 2 or H type 3/4. The H type 1 was expressed in goblet cells. Positive goblet cells expressing H type 1 were decreased in number progressively from the proximal colon to the rectum. In tumors, 4 (57%) of 7 cancer tissues of the proximal colon from secretors expressed no H type 1, whereas all 8 cancer tissues of the distal colon from secretors expressed H type 1. The aberrant expressions of H type 2 and H type 3/4 (47 and 67%, respectively) were found in cancer tissues from both the proximal and the distal colon. Tumors from nonsecretors did not express any H antigens. Our results suggested that the expression of H type 1 in the normal colon and the aberrant expressions of H type 2 and H type 3/4 in colon cancer tissues were regulated by FUT2-encoded Se type (1,2)fucosyltransferase. However, UEA-I-positive substance(s) rather than H type 2 were uniquely expressed throughout the normal colon and in colon cancers from both secretors and nonsecretors.     

Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor II stimulation of DNA synthesis in human fibroblasts

To investigate the role of insulin-like growth factor II in the control of DNA synthesis in human fibroblasts, dose-response curves for insulin-like growth factor I and II stimulation of [3H]thymidine incorporation were compared in the absence and presence of alpha IR-3, a highly specific monoclonal antibody directed against the type I insulin-like growth factor receptor. Specific binding of [125I]insulin-like growth factor I to human fibroblast monolayer cultures was inhibited 60-70% in the presence of alpha IR-3. alpha IR-3 had no effect on [125I]insulin-like growth factor II binding to human fibroblasts. However, alpha IR-3 inhibited both insulin-like growth factor I and II stimulated [3H]thymidine incorporation. These data indicate that the type II insulin-like growth factor receptor does not function as a transducer of insulin-like growth factor II's mitogenic effect in human fibroblasts.     

Synthesis of mono- and di-fucosylated type I Lewis blood group antigens by Helicobacter pylori.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

Modulation of a major 30-kDa skeletal muscle protein by thyroid hormone

Thyroidectomy results in the transformation of type II fibres to type I in rat soleus muscle. In vitro translations containing polyribosomes indicate that the template activity of mRNA coding for a 30-kDa protein is increased in hypothyroid (6 months) rats. The cellular content of this protein is also increased in hypothyroid rats. The in vitro synthesis of the 30-kDa protein is not observed in thyroidectomized (10 weeks) rats that have been treated with triiodothyronine. The synthesis and accumulation of this protein are directly related to the proportion of type I fibres in rat skeletal muscle and appear to be modulated by thyroid hormone.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Molecular cloning and functional expression of a novel Helicobacter pylori alpha-1,4 fucosyltransferase

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Galb-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Galb-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori alpha-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75-87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (k(cat)/Km = 229 mM(-1) s(-1)) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereof.     

Expression of collagen type I and type II in consecutive stages of human osteoarthritis

In normal hyaline cartilage the predominant collagen type is collagen type II along with its associated collagens, for example, types IX and XI, produced by normal chondrocytes. In contrast, investigations have demonstrated that in vitro a switch from collagen type II to collagen type I occurs. Some authors have detected collagen type I in osteoarthritic cartilage also in vivo, especially in late stages of osteoarthritis, while others have not. In the light of these diverging results, we have attempted to elucidate which type of collagen, type I and/or type II, is synthesized in the consecutive stages of human osteoarthritis. We performed in situ hybridization and immunohistochemistry with cartilage tissue samples from patients suffering from various stages of osteoarthritis. Furthermore, we quantitated our results on the gene expression of collagen type I and type II with the help of real-time PCR. We found that with the progression of the disease not only collagen type II, but also increasing amounts of collagen type I mRNA were produced. This supports the conclusion that collagen type I gradually becomes one of the factors involved in the pathogenesis of osteoarthritis.     

Production of alloantisera against class II bovine lymphocyte antigens (BoLA) by cross-immunization between class I matched cattle

This paper describes the production of alloantisera directed against bovine major histocompatibility complex (MHC) (BoLA) class II antigens in animals whose MHC phenotypes had been defined by one dimensional isoelectric focusing. Animals of closely matched BoLA class I types were selected by serology and subsequently typed for class I and class II by 1D-IEF of immunoprecipitated antigens. Those with similar class I type by both methods, but differing at the class II locus, were chosen for reciprocal immunization. Cross-immunization was by two skin implantations 6 weeks apart. The resulting antisera showed low titre after the first immunization and elevated titre 3 weeks after the second immunization. The sera reacted strongly with cells expressing specific BoLA class II antigens. The pattern of reactivity correlated well with IEF class II typing on a panel of animals representing all of the class II IEF types present in the Friesian population.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Lymphocyte transformation to connective tissue antigens in adult and juvenile rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus, and a nonarthritic control population

Transformation of peripheral blood lymphocytes after exposure to connective tissue antigens was measured in patients with adult (n = 35) and juvenile rheumatoid arthritis (n = 34), osteoarthritis (n = 21), ankylosing spondylitis (n = 15), and systemic lupus erythematosus (n = 26) and in control subjects (n = 36). The connective tissue antigens included homologous cartilage-type proteoglycan, cyanogen bromide-derived peptides of type I, II, and III collagens, and type I and II helical collagens. Lymphocyte transformation was not detected in the osteoarthritic and control groups, with one exception. Sensitization to at least one connective tissue antigen was detected in approximately one-third of the rheumatoid arthritic and lupus patients and in one-quarter of the juvenile rheumatoid patients. In ankylosing spondylitis, positive responses occurred to proteoglycan in 20% of patients tested but never to collagens or peptides. Sensitivity to proteoglycan was detected only in ankylosing spondylitis except for one patient with juvenile rheumatoid arthritis. In patients with systemic lupus erythematosus and both forms of rheumatoid arthritis, lymphocyte transformation was usually more frequently detected to peptides than to the helical collagens. In adult rheumatoid arthritis, type II peptides elicited an elevated number of responses (14%) as did type I (9%) and III (8%) peptides to lesser degrees. Responses to type I (4%) and II (4%) helical collagens were infrequent. Rheumatoid arthritic patients usually exhibited sensitivity to only one antigen and lymphocyte transformation was often detected when the arthritis was improving. In juvenile rheumatoid arthritis, lymphocyte transformation was detected to peptides of type I (16%), II (9%), and III (29%) collagens and to helical type I (12%) and II (8%) collagens. In systemic lupus erythematosus, sensitization was detected to peptides of type I (13%), II (20%), and III (14%) collagens and to helical type I collagen (18%) but not type II collagen. Simultaneous sensitivity to several antigens often occurred in both systemic lupus erythematosus and juvenile rheumatoid arthritis. Examination of individual patients in all three rheumatic disease groups revealed that immune sensitivity developed to collagen peptides rather than to the helical molecules, particularly in the case of type II collagen. Thus, some patients with inflammatory arthritis exhibit immune responses to connective tissue components which are, as a group, characteristic for each type of arthritis. These responses, which were not obviously associated with disease activity, may develop as a result of inflammation or trauma which destroys connective tissue and exposes molecules, in either a native or degraded state, to cells of the immune system. Expression of sensitivity to these tissue antigens may contribute to the chronicity of the inflammatory arthritides.     

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.     

Molecular analysis to FUT-1, 2, 3 and ABO genotyping may instead of serological typing to diagnose para-Bombay in Chinese

The aim of this study was to evaluate the consistency between serotyping and molecular analysis in Chinese with para-Bombay. The molecular analysis of gene fragments in FUT-1, FUT-2, FUT-3 and ABO genotyping and serotyping were used including a saliva test to examine the A, B, H substance and an absorption elution test to examine the A, B, H; and further routine tests including ABO, H and Lewis phenotype. From eleven samples with anti-H negative, 10 samples were confirmed with para-Bombay by sequencing to FUT-1, from which six samples were 547-548delAG, three samples were 880TT deletion, one sample was 35C>T and one sample was 649G>T heterozygous (h7, China) as carrier. The sequencing to FUT-2 confirmed 357C>T in 11 samples, meaning H, A and B substance was secreted in saliva except for one sample which occurred 385A>T (I129F) heterozygous, which is a weak secretor. The FUT-3 sequence result demonstrated four samples with heterozygous mutations to 59T>G (L20R) combined with 508G>A (G170S) and seven samples without mutations in FUT-3 gene fragment same as reference. The consistency between sequencing with FUT-1/FUT-2 and serotyping by anti-H reported an identical result, except for one sample, which interestingly showed the H/h7 carrier with serotyping negative to anti-H. The result of sequencing with FUT-2/FUT-3 and Lewis phenotyping also reported a complete consistency. The saliva test to A, B, H substance and absorption elution test examining the A, B, H antigens on the surface of red blood cells completely matched the ABO exon 6, 7 sequence results. The sequencing of FUT-1, FUT-2, FUT-3 and ABO exon 6, 7 may become a useful tool to confirm the para-Bombay blood type.