Mucin core O-glycosylation is modulated by neighboring residue glycosylation status. Kinetic modeling of the site-specific glycosylation of the apo-porcine submaxillary mucin tandem repeat by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases T1 and T2

The influence of peptide sequence and environment on the initiation and elongation of mucin O-glycosylation is not well understood. The in vivo glycosylation pattern of the porcine submaxillary gland mucin (PSM) tandem repeat containing 31 O-glycosylation sites (Gerken, T. A., Gilmore, M., and Zhang, J. (2002) J. Biol. Chem. 277, 7736-7751) reveals a weak inverse correlation with hydroxyamino acid density (and by inference the density of glycosylation) with the extent of GalNAc glycosylation and core-1 substitution. We now report the time course of the in vitro glycosylation of the apoPSM tandem repeat by recombinant UDP-GalNAc:polypeptide alpha-GalNAc transferases (ppGalNAc transferase) T1 and T2 that confirm these findings. A wide range of glycosylation rates are found, with several residues showing apparent plateaus in glycosylation. An adjustable kinetic model that reduces the first-order rate constants proportional to neighboring glycosylation status, plus or minus three residues of the site of glycosylation, was found to reasonably reproduce the experimental rate data for both transferases, including apparent plateaus in glycosylation. The unique, transferase-specific, positional weighting constants reveal information on the peptide/glycopeptide recognition site for each transferase. Both transferases displayed high sensitivities to neighboring Ser/Thr glycosylation, whereas ppGalNAc T2 displayed additional high sensitivities to the presence of nonglycosylated Ser/Thr residues. This is the first demonstration of the ability to model mucin O-glycosylation kinetics, confirming that under the appropriate conditions neighboring glycosylation status can be a significant factor modulating the first step of mucin O-glycan biosynthesis.     

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1, 3-sulphate-Gal{beta}1-3GalNAc{alpha}-R

A novel sulphotransferase (sulpho-T) activity from rat colonicmucosa was characterized using O-glycan core 1 substrate, Galß1-3GalNAc     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-alpha-galactosaminyl-R beta 3-galactosyltransferase

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc alpha-R beta 3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of beta 3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the beta 3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain beta 3- and beta 6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc beta 3GalNAc alpha-R) and core 4 [GlcNAc beta 6(GlcNAc beta 3)GalNAc alpha-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with beta 3-Gal-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, the 15-residue peptide PPAHGVTSAPDTRPA (PPA15) from the MUC1 protein core and its analogue PPA15(T7), glycosylated with alpha-N-acetylgalactosamine on Thr7, were prepared and investigated by NMR spectroscopy. The peptide contains both the GVTSAP sequence, which is an effective substrate for GalNAc-T1 and -T3 transferases, and the PDTRP fragment, which is a well-known immunodominant epitope recognized by several anti-MUC1 monoclonal antibodies. Useful structural results were obtained in water upon decreasing the temperature to 5-10 degrees C. The sugar attachment slightly affected the conformational equilibrium of the peptide backbone near the glycosylated Thr7 residue. The clustering of low-energy conformations for both PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed structural similarities between glycosylated and nonglycosylated peptides. For the GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA15(T7) conformers showed distinct structural propensities of the peptide backbone specific for either the nonglycosylated or the glycosylated peptide. The peptide backbone of the APDTRP fragment, which is a well-known immunodominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the peptide backbone is formed by consecutive inverse gamma-turn conformations partially stabilized by hydrogen bonding. A comparison of the solution structure of the APDTRP fragment with a crystal structure of the MUC1 peptide antigen bound to the breast tumor-specific antibody SM3 demonstrated significant structural similarities in the general shape.     

S-adenosyl-L-methionine activates actinorhodin biosynthesis by increasing autophosphorylation of the Ser/Thr protein kinase AfsK in Streptomyces coelicolor A3(2)

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolor A3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsK plays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KN mutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitro autophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolor M130 by increasing the phosphorylation of protein kinase AfsK.     

(1)H NMR studies of hydroxy protons of the V[beta-Gal(1-->3)-alpha-GalNAc(1-->O)]THPGY glycopeptide.

The hydroxy protons of the disaccharide moiety in the glycopeptide Val-[beta-Gal(1-->3)-alpha-GalNAc(1-->O)]-Thr-His-Pro-Gly-Tyr (1) have been investigated in aqueous solution using (1)H NMR spectroscopy. The chemical shifts (delta), coupling constants ((3)J(CH,OH)), temperature coefficients (d delta/dT), exchange rates (k(ex)), and NOEs have been measured. The data show that the O(2')H of Gal has a reduced contact with water due to steric interference caused by the 2-acetamido group of GalNAc. No interaction, in terms of hydrogen bonding exists between the disaccharide and the peptide moieties, but the rotation around the sugar-peptide linkage is restricted.     

Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation

Invasion of the basement membrane is believed to be a critical step in the metastatic process. Melanoma cells have been shown previously to bind distinct triple-helical regions within basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models of the type IV collagen alpha1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and (b) analyze the results of single-site glycosylation on melanoma cell recognition. Receptor identification was achieved by a combination of methods, including (a) cell adhesion and spreading assays using triple-helical alpha1(IV)1263-1277 and an Asp(1266)Abu variant, (b) inhibition of cell adhesion and spreading assays, and (c) triple-helical alpha1(IV)1263-1277 affinity chromatography with whole cell lysates and glycosaminoglycans. Triple-helical alpha1(IV)1263-1277 was bound by melanoma cell CD44/chondroitin sulfate proteoglycan receptors and not by the collagen-binding integrins or melanoma-associated proteoglycan. Melanoma cell adhesion to and spreading on the triple-helical alpha1(IV)1263-1277 sequence was then compared for glycosylated (replacement of Lys(1265) with Hyl(O-beta-d-galactopyranosyl)) versus non-glycosylated ligand. Glycosylation was found to strongly modulate both activities, as adhesion and spreading were dramatically decreased due to the presence of galactose. CD44/chondroitin sulfate proteoglycan did not bind to glycosylated alpha1(IV)1263-1277. Overall, this study (a) is the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, (b) provides a rare example of an apparent unfavorable interaction between carbohydrates, and (c) suggests that sugars may mask "cryptic sites" accessible to tumor cells with cell surface or secreted glycosidase activities.     

Nitric oxide (NO) and cartilage metabolism: NO effects are modulated by superoxide in response to IL-1

Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.     

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.     

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.     

Anxiogenic and antinociceptive effects induced by corticotropin-releasing factor (CRF) injections into the periaqueductal gray are modulated by CRF1 receptor in mice

Chemical or electrical stimulation of the dorsal portion of the midbrain periaqueductal gray (dPAG) produces anxiogenic and antinociceptive effects. In rats, chemical stimulation of dPAG by local infusion of the neuropeptide corticotropin-releasing factor (CRF) provokes anxiogenic effects in the elevated plus-maze test (EPM). CRF also produces antinociception when injected intracerebroventricularly in rats, however it remains unclear whether this response is also observed following CRF injection into the dPAG in mice. Yet, given that there are CRF1 and CRF2 receptor subtypes within the PAG, it is important to show in which receptor subtypes CRF exert its anxiogenic and antinociceptive effects in the dPAG. Here, we investigated the role of these receptors in the anxiogenic (assessed in the EPM) and antinociceptive (assessed by the Formalin test: 2.5% formalin injection into the right hind paw) effects following intra-dPAG infusion of CRF in mice. The results show that intra-dPAG injections of CRF (75 pmol/0.1 μl and 150 pmol/0.2 μl) produced dose-dependent anxiogenic and antinociceptive effects. In addition, local infusion of NBI 27914 (5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)-aminopyridine; 2 nmol/0.2 μl), a CRF1 receptor antagonist, completely blocked both the anxiogenic and antinociceptive effects induced by local infusion of CRF, while that of antisauvagine 30 (ASV30; 1 nmol/0.2 μl), a CRF2 receptor antagonist, did not alter the CRF effects. Present results are suggestive that CRF1 (but not CRF2) receptors play a crucial role in the anxiogenic and antinociceptive effects induced by CRF in the dPAG in mice.     

APP independent and dependent effects on neurite outgrowth are modulated by the receptor associated protein (RAP)

Amyloid precursor protein (APP) and its secreted form, sAPP, contribute to the development of neurons in hippocampus, a brain region critical for learning and memory. Full‐length APP binds the low‐density lipoprotein receptor‐related protein (LRP), which stimulates APP endocytosis. LRP also contributes to neurite growth. Furthermore, the receptor associated protein (RAP) binds LRP in a manner that blocks APP–LRP interactions. To elucidate APP contributions to neurite growth for full‐length APP and sAPP, we cultured wild type (WT) and APP knockout (KO) neurons in sAPPα and/or RAP and measured neurite outgrowth at 1 day in vitro. Our data reveal that WT neurons had less axonal outgrowth including less axon branching. RAP treatment potentiated the inhibitory effects of APP. KO neurons had significantly more outgrowth and branching, especially in response to RAP, effects which were also associated with ERK2 activation. Our results affirm a major inhibitory role by full‐length APP on all aspects of axonal and dendritic outgrowth, and show that RAP–LRP binding stimulated axon growth independently of APP. These findings support a major role for APP as an inhibitor of neurite growth and reveal novel signaling functions for LRP that may be disrupted by Alzheimer's pathology or therapies aimed at APP processing.     

Corticotropin-releasing factor (CRF)-induced behaviors are modulated by intravenous administration of teneurin C-terminal associated peptide-1 (TCAP-1)

The teneurin C-terminal associated peptides (TCAP) are a recently discovered family of bioactive peptides that can attenuate aspects of the behavioral stress responses of rats. Because TCAP has some structural similarity to the corticotropin-releasing factor (CRF) family of peptides, and modulates elements of the stress response, TCAP may act to modulate CRF actions in vivo. This hypothesis was tested by investigating anxiety-related behaviors in male rats following repeated intravenous (IV) TCAP-1 administration with either an acute intracerebroventricular (ICV) or IV CRF challenge. TCAP-1 alone did not affect behavioral responses significantly, however did significantly affect CRF-regulated behaviors depending on CRF's mode of injection. In both the elevated plus-maze and the open field tests, TCAP-1 had an anxiolytic effect on ICV CRF responses as indicated by decreased stretched-attend postures in the elevated plus maze (p<0.05), and increased center time and center entries in the open field (p<0.05). However, prior TCAP-1 treatment has an anxiogenic effect on the IV CRF-induced behaviors (decreased center entries and total distance in the open field (p<0.05)). TCAP-1's actions are not mediated through acute changes in glucocorticoid levels and may occur via a central action in the brain. A fluorescently (FITC)-labeled TCAP-1 analog was IV-administered to investigate whether IV TCAP-1 has the potential to regulate central mechanisms by crossing the blood-brain barrier. FITC-TCAP-1 was detected in blood vessels and fibers in the brain indicating that uptake into the brain is a possible route for its interaction with CRF and its receptors. Thus, TCAP may modulate CRF-associated behaviors by a direct action in the CNS.     

Cytotoxic effects of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCHQ) on liver cells are modulated by antioxidants

The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.     

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.     

Carbohydrate recognition factors of a Talpha (Galbeta1-->3GalNAcalpha1-->Ser/Thr) and Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha

Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes > monomeric Talpha and simple clustered Tn > monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.     

Kaempferol-3-O-glucosyl(1-2)rhamnoside from Ginkgo biloba and a reappraisal of other gluco(1-2, 1-3 and 1-4)rhamnoside structures.

A kaempferol-3-O-glucorhamnoside from Ginkgo biloba is defined as the 3-O-alpha-L-[ beta-D-glucopyranosyl(1-2)rhamnopyranoside] on the basis of 2D NMR evidence. Complete assignments of the 1H and 13C NMR spectra of this compound and of its known p-coumaroyl derivative are presented for the first time. The NMR distinctions of 1-2, 1-3 and 1-4 linked glucopyranosylrhamnopyranosides are discussed and indicate (i) that the 13C NMR assignments for one published gluco(1-3)rhamnoside are in need of modification, (ii) that the published structure of hordenine-O-[6-O-t-cinnamoyl-beta-glucosyl(1-4)-alpha-rhamnoside] from Selaginella doederleinii is not distinguished from the 1-3 linked glucorhamnoside structure, and (iii) that the 8-prenylkaempferol-3-O-[glucosyl(1-4)rhamnoside]-7-O-glucoside and the equivalent 4'-O-methylated xylosyl(1-4)rhamnoside from Epimedium pubescens and E. washanense, respectively, are (1-2)-linked.     

Tetraspanin CD151 regulates glycosylation of (alpha)3(beta)1 integrin

The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.