Lectin Peanut Agglutinin Research Articles

Investigation of the interaction between peanut agglutinin and synthetic glycopolymeric multivalent ligands

The interaction between synthetic glycoplymers bearing beta-D-galactose side groups and the lectin peanut agglutinin (PNA) was investigated by UV-difference spectroscopy and isothermal titration calorimetry (ITC). UV-difference spectroscopy indicated that the polymer-lectin interaction was stronger than that between PNA and either the corresponding monomer, D-galactose or D-lactose. The thermodynamics of binding (K, DeltaG, DeltaH, DeltaS and n) were determined from ITC data by fitting with a two-site, non-cooperative binding model. It was found that the glycopolymer displayed around a 50 times greater affinity for the lectin than the parent carbohydrate, and around 10 times greater than the monomer, on a valency-corrected basis. Binding was found to be entropically driven, and was accompanied by aggregation and precipitation of protein molecules. Furthermore, interesting differences between polymers prepared either from deacetylated monomers, or by deacetylation of pre-formed polymers, were found.

Characterization of the Alzheimer's Disease-associated CLAC Protein and Identification of an Amyloid β-Peptide-binding Site

Amyloid beta-peptide (Abeta) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with Abeta in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Abeta plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Abeta. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Abeta-(1-40) and Abeta-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Abeta. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Abeta deposits.

An Inhibitor of O-Glycosylation Induces Apoptosis in NIH3T3 Cells and Developing Mouse Embryonic Mandibular Tissues

The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.

Retinal changes after macular translocation with 360-Degree retinotomy in monkey eyes

PurposeTo determine the morphologic and functional changes of the fovea and retina of monkey eyes after macular translocation with 360-degree retinotomy. DesignExperimental study. MethodsThe retinas of eight monkey eyes were surgically translocated with a 360-degree retinotomy with procedures similar to those used on human eyes. At 1, 2, and 3 months after the surgery, the six eyes that had successful surgery were studied by light and transmission electron microscopy, terminal deoxynucleotidyl transferase (TdT)-dNTP terminal nick-end labeling (TUNEL) assay, and immunohistochemistry with peanut agglutinin (PNA) lectin and glial fibrillary acidic protein (GFAP). Retinal physiology was assessed by scotopic and photopic electroretinograms (ERGs). ResultsThe fovea was successfully translocated approximately 30 to 40 degrees superiorly in six eyes. The translocated macula and fovea had a normal layered architecture with no TUNEL-positive cells, minimal misalignment of the outer segments, and strong immunoreactivity to GFAP. The mean amplitudes of the scotopic and photopic b-waves were significantly reduced at 1 month after the surgery, and there was only a slight recovery at 3 months. No significant changes were observed in the mean implicit times after the surgery. ConclusionThese findings indicate that macular translocation surgery with 360-degree retinotomy results in minimal morphologic alterations but significant depression of electrophysiologic function.

Glomerulocystic Kidney Disease in a Belgian Malinois Dog: An Ultrastructural, Immunohistochemical, and Lectin-binding Study

Renal cysts in the cortex of a juvenile Belgian Malinois dog with acute renal failure were studied by means of light, scanning and transmission electron microscopy, immunohistochemistry for intermediate filaments, and binding for wheat germ agglutinin (WGA), peanut agglutinin (PNA), and Maclura pomiferaagglutinin (MPA) lectins to determine the morphological and histochemical features of the epithelial cells of these cysts. The cysts were renal corpuscles with expanded urinary space. Glomerular tufts were small with poorly developed capillary loops and increased mesangial matrix. Continuity with the proximal tubule was evident in some cystic glomeruli. Two cell types lined Bowman's capsule. One was squamous with a central cilium and microvilli. The other had morphological and histochemical features of immature podocytes (parietal podocytes). These cells were round and protruded into the urinary space; they had thick cytoplasmic projections that resembled foot processes of podocytes, microvilli, and filtration slits. The parietal podocytes expressed vimentin and cytokeratins and had affinity for WGA as do normal immature podocytes. These features suggest that the parietal podocytes are derived by metaplasia of the parietal cells. The basement membrane of Bowman's capsule was irregularly thickened and showed multifocal glycosylation changes with lectin histochemistry (WGA, PNA, MPA) in areas adjacent to the parietal podocytes. Histologic and ultrastructural findings in this dog are consistent with glomerulocystic kidney disease. This is the second report of canine glomerulocystic kidney disease. Features are similar to those of the human counterpart, but it is unclear whether genetic defects cause the disease in the dog. The presence of parietal podocytes in all cysts suggests that abnormal differentiation may play an important role in the pathogenesis of this type of polycystic kidney disease.

Core 1 Glycans on α-Dystroglycan Mediate Laminin-induced Acetylcholine Receptor Clustering but Not Laminin Binding

Although unique O-linked oligosaccharides on alpha-dystroglycan are important for binding to a variety of extracellular ligands, the function(s) of more generic carbohydrate structures on alpha-dystroglycan remain unclear. Recent studies suggest a role for glycoconjugates bearing the core 1 disaccharide Galbeta(1-3)GalNAc in acetylcholine receptor (AChR) clustering on the surface of muscle cells. Here, we report experiments demonstrating that the core 1-specific lectin jacalin almost completely abrogated laminin-induced AChR clustering in C2C12 myotubes and that alpha-dystroglycan was the predominant jacalin-binding protein detected in C2C12 myotube lysates. Although jacalin likely inhibited laminin-induced AChR clustering by directly binding to alpha-dystroglycan, jacalin had no effect on laminin binding to the myotube surface or to alpha-dystroglycan. Like jacalin, peanut agglutinin lectin also binds the core 1 disaccharide but not when it is terminally sialylated as expressed on alpha-dystroglycan. We show that C2C12 alpha-dystroglycan bound to peanut agglutinin only after digestion with neuraminidase. Simultaneous treatment of myotubes with neuraminidase and endo-O-glycosidase diminished alpha-dystroglycan binding to peanut agglutinin and inhibited neuraminidase-induced AChR clustering. We conclude that sialylated core 1 oligosaccharides of alpha-dystroglycan are important for laminin-induced AChR clustering and that their function in this process is distinct from the established role of alpha-dystroglycan oligosaccharides in laminin binding.

Glycophorin A requirement for expression of O-linked antigens on the erythrocyte membrane.

Glycophorin A (GPA) has a large number of sialic acid-containing oligosaccharide chains. GPA is highly conserved among vertebrates, mice with a GPA deletion have not been reported and GPA's physiologic role remains uncertain. GPA-/- homozygotes were obtained by intercrossing GPA+/- heterozygotes based on Mendelian genetics. The amount of O-linked oligosaccharide chains in the erythrocyte membrane of GPA-/- mice decreased to 60% compared to that of the wild-type mice. Flow cytometry and Western blot analysis revealed that the TER antigen that is associated with GPA on the erythrocyte membrane was totally abrogated from the cell surface in GPA-/- mice. Several glycoproteins that were detected with peanut agglutinin (PNA), a lectin that recognizes O-linked oligosaccharide chains, were absent from the GPA-/- erythrocyte membrane. Erythrocytes lacking GPA were more sensitive to hypo-osmotic stress than wild-type erythrocyte. GPA-/- mice show apparently normal phenotypes at least during the early generations. The disappearance of many glycoproteins recognized by PNA lectin on the GPA-/- erythrocyte membrane proteins suggests that GPA has an essential role in the expression of O-linked antigens on the erythrocyte membrane protein. These interactions of GPA and other glycoproteins may contribute to maintaining the physical strength of the erythrocyte membrane.

PyV-mT-induced parotid gland hyperplasia as detected by altered lectin reactivity is not modulated by inducible nitric oxide deficiency

The parotid gland was one of the first organs recognised to be sensitive to the transforming effects of polyomavirus. This study examines parotid gland pathology in mice expressing the polyomavirus middle T (PyV-mT) under the control of the mouse mammary tumour virus long terminal repeat (MMTV-LTR) to (1) demonstrate the utility of this model for studying premalignant disease; (2) identify early lesions by lectin staining and (3) determine effects of the inducible nitric oxide synthase (iNOS) in modulating tumorigenesis. The middle T oncogene is expressed in the parotid glands in addition to the mammary glands and results in the formation of parotid hyperplasias in 100% of transgenic female mice. These hyperplasias have the features of intraepithelial neoplasia including hypertrophic cells with prominent nucleoli and abnormal mitoses. Focal areas of parotid hyperplasia can be identified using peanut agglutinin (PNA), a lectin that recognises the tumour associated T antigen. In contrast to normal parotid gland, areas of hyperplasia do not bind PNA. Mice deficient in iNOS, an enzyme implicated in the promotion of tumorigenesis, were bred into the PyV-mT model. The loss of iNOS did not impact on the number or size of parotid gland hyperplasias, suggesting that NO produced by this enzyme is not a key regulator of PyV-mT-induced parotid gland hyperplasia. The consistent development of parotid hyperplasias in PyV-mT mice and clear identification of these lesions by loss of PNA lectin reactivity provides a useful model for studying early molecular changes in parotid tumorigenesis.

Effect of sperm preparation method on in vitro fertilization in pigs.

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

Effect of sperm preparation method on in vitro fertilization in pigs

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

Characterization of calbindin-positive cones in primates

The aim of this study is to characterize calbindin-positive photoreceptors and their opsin content in the retina of nocturnal prosimians ( Microcebus murinus), New World monkeys ( Callithrix jacchus), Old World monkeys ( Macaca fascicularis), and humans. To identify the calbindin and opsin content of cones, combined multiple labeling with different fluorescent probes, antibodies directed against calbindin, short, and mid–long wavelength opsins, and lectin peanut agglutinin cytochemistry were used. With the exception of Microcebus, calbindin is present in the cones of all primates but is absent from rods. The distribution of calbindin is similar in human and macaque cones, with dense label in the inner segment, cell body, axon and cone pedicle. Cones in marmoset also show dense staining in the cell body, axon and pedicle but only light label in the inner segment. Primate cone outer segments do not contain calbindin. In the primates studied, three patterns of calbindin and opsin localization are observed. In macaque and marmoset all short and mid–long wavelength cones contain calbindin. In humans, all mid–long wavelength cones contain calbindin whereas all short wavelength cones are devoid of calbindin as confirmed by confocal microscopy. In the nocturnal prosimian Microcebus none of the mid–long or short wavelength cones contain calbindin. In addition to primates, calbindin is absent in cones of other nocturnal species but is present in cones of diurnal species suggesting a difference in the role of calbindin possibly related to the adaptational states or other photoreceptor properties.

Fate of the acrosome in ooplasm in pigs after IVF and ICSI.

ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.

Lectin-erythrocyte interaction with external transmembrane glycophorin saccharides controlling membrane internal cytoskeleta.

Human red blood cell (RBC, erythrocyte) membranes have internal protein skeletons that govern the cells' distinctive discocyte-echinocyte morphology (shape) changes, seen in conventional microscopy. Glycophorin, the cell's transmembrane protein, presents all of its saccharides outside the cell. The protein sector of glycophorin is linked inside to the RBC cytoskeleton, enabling lectins binding to the external saccharides to gain profound control over internal cytoskeleton behavior, expressed by governance of the visibly seen cell shape. Critical lectin binding stoichiometries ((125)I-labeled lectins) equate to the number of glycophorin monomers per RBC, 7 x 10(5) copies/cell. Wheat germ agglutinin lectin (sialic acid specific) binds to glycophorin's outermost (exo) saccharides and exerts tight control over the cell's morphology. Removal of sialic acid groups (desialation) exposes the endosaccharides of glycophorin, enabling peanut agglutinin and Osage orange lectins to gain equally tight control over the RBC's morphology behavior in simple stoichiometric ratios, bound lectin molecules/glycophorin receptor. Thus, lectin specificities for saccharides are sharply in register with the glycophorin external saccharide composition, the sequence along the chains, and the number of copies of protein (stoichiometry). These relationships were determined via RBC shape change equilibria and also via shape change rates. Rate data are somewhat laborious to determine, but are exquisitely sensitive to lectin specificities and in very small lectin concentrations. Both classes of data enable these interactions to be analyzed in lectin and RBC concentrations approximately 100-fold smaller than agglutinating levels.

Dynamic regulation of T cell immunity by CD43.

During a viral response, Ag-specific effector T cells show dramatically increased binding by the mAb 1B11 and the lectin peanut agglutinin (PNA). We investigated the contribution of CD43 expression to 1B11 and PNA binding as well as its role in generation and maintenance of a CD8 T cell response. Analysis of CD43(-/-) mice revealed no increased 1B11 binding and reduced PNA binding on virus-specific CD8 T cells from -/- mice compared with +/+ mice. Furthermore, we examined the role of CD43 in the kinetics of an immune response. We show that CD43 expression modestly effects generation of a primary virus-specific CD8 T cell response in vivo but plays a more significant role in trafficking of CD8 T cells to tissues such as the brain. More interestingly, CD43 plays a role in the contraction of the immune response, with CD43(-/-) mice showing increased numbers of Ag-specific CD8 T cells following initial expansion. Following the peak of expansion, Ag-specific CD8 T cells from -/- mice show similar proliferation but demonstrate increased Bcl-2 levels and decreased apoptosis of Ag-specific effector CD8 T cells in vitro. Consistent with a delay in the down-modulation of the immune response, following chronic viral infection CD43(-/-) mice show increased morbidity. These data suggest a dynamic role of CD43 during an immune response: a positive regulatory role in costimulation and trafficking of T cells to the CNS and a negative regulatory role in the down-modulation of an immune response.

Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar structures by Arachis hypogaea (peanut agglutinin) lectin.

Epimastigote culture forms of different isolates of Trypanosoma cruzi from different mammal hosts, humans, and vectors were tested with FITC-conjugated peanut agglutinin lectin (PNA-FITC). The parasites maintained in axenic medium, liver infusion tryptose. were evaluated by flow cytometric analyses; whereas T. cruzi I (Tcl), which is associated with the sylvatic transmission cycle, was labeled in high percentages with PNA (88-99.2%), T. cruzi II (TcII) (parasites associated with domiciliar cycle) and T. cruzi, zymodeme 3 (Tc/Z3) (also associated with the sylvatic cycle) were labeled in low percentages (TcII, 0-26% and Tc/Z3, 0-12.6%). It was demonstrated that it is possible to differentiate the 2 main T. cruzi subpopulations, TcI and TcII, using Arachis hypogaea. These results also showed a higher variability in TcII in terms of PNA binding.

Papular xanthoma: a clinicopathological study of 10 cases.

Papular xanthoma (PX) is one of several clinicopathologic variants of normolipemic cutaneous non-Langerhans cell histiocytoses (n-LCH). PX represents a monomorphous reaction pattern of n-LCH characterized by the presence of predominantly xanthomatized macrophages. The purpose of this study was to identify the clinical, histological and immunohistochemical characteristics of PX. A series of 10 cases of PX was identified and the results compared with the other histologic subtypes, namely the polymorphous and the remaining other monomorphous reaction patterns in n-LCH. In this clinicopathologic study, papular xanthoma presented clinically mainly as solitary papule, with a male to female ratio of4 : 1, in an age range from 13 to 57 years and a biphasic occurrence: in the young adolescence and middle ages. It was predominantly located on the trunk, the extremities, and rarely on the head. Clinically, PX was described as xanthoma, 'cutaneous tumor', but also as atheroma, keloid, histiocytoma, Spitz's nevus or clear cell acanthoma. Histology showed moderately well circumscribed exoendophytic papules with a regular epidermis and a dense infiltration of xanthomatized macrophages interspersed by numerous Touton type giant cells. Immunohistochemically mono- and multinucleated macrophages were consistently positive with KiM1p; while only giant cells were labeled with KP1 (CD68), the reactivity with HAM 56 was much more variable. Up to 50% of the xanthomatized cells labeled positive for the lectin peanut agglutinin. In one case the xanthomatized cells stained positive for CD34. Staining for factor XIIIa and CD1a were negative. This series confirms PX as a rare, but distinguished clinicopathologic entity in the spectrum of n-LCH of the skin.

Photoswitchable multivalent sugar ligands: synthesis, isomerization, and lectin binding studies of azobenzene-glycopyranoside derivatives.

Coating of azobenzene chromophore with multivalent sugar ligands has been accomplished. Such sugar coating allows the study of the isomerization properties of this chromophore in aqueous solutions. The predominantly cis-isomer-containing photostationary state (PS) mixture of these azobenzene derivatives is found to be stable for hours. The rate constants for their isomerization, as well as the Arrhenius activation energies, are determined experimentally. An assessment of the lectin binding properties of the lactoside bearing isomeric azobenzene derivatives, by isothermal calorimetric methods, reveals the existence of an unusual cooperativity in their binding to lectin peanut agglutinin. Thermodynamic parameters evaluated for the trans and the PS mixture are discussed, in detail, for the lactoside bearing bivalent azobenzene derivative.

Extracellular matrix glycoproteins inhibit neurite production by cultured neurons.

We have analyzed the role of extracellular matrix glycoproteins in the formation of a bipolar outgrowth pattern of identified leech neurons in culture. Adult anterior pagoda (AP) neurons cultured on the inner surface of the ganglion capsules that surround central nervous system, generate two processes oriented in opposite directions. This pattern differs from those produced by these neurons cultured on other substrates, and is similar to the pattern of developing AP neurons at embryonic day 10. We used different lectins to identify subsets of glycoproteins in the extracellular matrix (ECM) of the capsules and to study their contribution to the formation of the bipolar outgrowth pattern. ECM glycoproteins binding to peanut agglutinin (PNA) or Galanthus nivalis aglutinin (GNA) lectins were detected in ganglion capsules and in ganglion extracts that had been separated by electrophoresis and blotted to nitrocellulose membranes. Four protein bands bound to PNA lectin and six other bands, including laminin subunits, bound to GNA lectin. Other lectins failed to recognize any of the proteins. For AP neurons cultured on capsules, addition of PNA lectin to the culture medium produced a dose-dependent increase in the number of primary neurites without affecting their shape, length or number of branch points. However, PNA lectin used as substrate did not affect sprouting of AP neurons. Our results suggest that PNA-binding extracellular matrix glycoproteins regulate the formation of the bipolar pattern of AP neurons by inhibiting the formation of neurites.

Increased sialylation of polymeric lambda-IgA1 in patients with IgA nephropathy.

The mechanism of mesangial IgA deposition is poorly understood in IgA nephropathy (IgAN). Abnormal glycosylation of carbohydrate moieties in the hinge region of the IgA molecule has recently attracted much attention. In this report, we studied galactosylation and sialylation profiles in kappa- and lambda-IgA1 from patients with IgAN. Total serum IgA1 was isolated from patients with IgAN or healthy controls by jacalin-affinity chromatography. Six fractions of molecular weight (MW) 50-1,000 kDa were separated by fast protein liquid chromatography (FPLC). Four lectin-binding assays were used to study the sialylation and the presence of terminal galactose or N-acetylgalactosamine (GalNAc) in the O-linked carbohydrate moieties of kappa- or lambda-IgA1. Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) lectin recognize alpha(2,3)- and alpha(2,6)-linked sialic acid, respectively. Peanut agglutinin (PNA) and Helix aspersa (HA) lectin recognize terminal galactose and GalNAc, respectively. Reduced HA was demonstrated in macromolecular kappa or lambda-IgA1 (300-825 kDa) isolated from patients with IgAN (P < 0.05 compared with healthy controls). Lambda- but not kappa-IgA1 from patients with IgAN bound less to PNA (P < 0.05). The alpha(2,3)-linked sialic acid content in lambda- but not kappa-IgA1 of MW 150-610 kDa from patients was higher than that of controls (P < 0.005). The alpha(2,6)-linked sialic acid content in lambda-IgA1 (300-825 kDa) and kappa-IgA1 (150-610 kDa) from patients was also higher than that of controls. This unusual glycosylation and sialylation pattern of the lambda-IgA1 may have important implications for the pathogenesis of IgAN, as both the masking effect of sialic acid on galactose and the reduced galactosylation will hinder the clearance of macromolecular lambda-IgA1 by asialoglycoprotein receptor of hepatocytes. The negative charge from sialic acid may also favor mesangial deposition of macromolecular lambda-IgA1 in IgAN.

Regulated by Sialylation

As thymocytes mature, their cell-surface proteins change both in terms of which proteins are present and in the structure of the proteins present. Moody et al. investigated how changes in glycosylation of the CD8 cell-surface glycoprotein of thymocytes influence its interaction with major histocompatibility complex class I (MHCI) molecules. Peptide MHCI tetramers (pMHCIs) were used in combination with fluorescent cell sorting (FACS) analysis to show that immature thymocytes that are positive for both CD8 and CD4 (another cell-surface receptor), so-called "DP" thymocytes, interact with the pMHCIs. Mature thymocytes that only express CD8 or CD4 (so-called "SP" thymocytes) did not interact with the pMHCIs, despite the fact that the DP and CD8 SP thymocytes express the same amount of CD8α and β subunits. Biochemical analysis of CD8α and β from DP and SP thymocytes showed that the transition to SP led to a decrease in molecular heterogeneity of the β subunit and marked reduction in the ability of the β subunit to be affinity-purified by the lectin peanut agglutinin (PNA). Treatment of SP thymocytes with neuraminidase to remove sialic acid from O -glycans restored PNA and pMHCI binding, suggesting that sialylation of the β subunit is responsible for the change in MHCI binding capacity of the SP thymocytes. Analysis of pMHCI binding to thymocytes from a mouse lacking the sialyltransferase ST3Gal-1 gene showed that the SP thymoctyes bound pMHCIs and maintained a high PNA-binding character throughout maturation. The change in glycosylation and the resulting effect on MHC binding appear to influence CD8 thymic selection in that in 4 of 14 animals tested, the lack of the ST3 Gal-1 gene resulted in a decrease in the number of CD8-positive SP thymocytes. A. M. Moody, D. Chui, P. A. Reche, J. J. Priatel, J. D. Marth, E. L. Reinherz, Developmentally regulated glycosylation of the CD8αβ coreceptor stalk modulates ligand binding. Cell 107 , 501-512 (2001). [Online Journal]