Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Fluorogenic substrates of glycogen debranching enzyme for assaying debranching activity

Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (B3), Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B4), Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5), Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B6), Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B7), and Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

The sugar chains of cold-insoluble globulin. A protein related to fibronectin.

Cold-insoluble globulin isolated from bovine plasma contains six asparagine-linked sugar chains in 1 molecule (a dimeric form). These sugar chains were released from the polypeptide backbone by hydrazinolysis and labeled by reduction with NaB[3H]4. Most of these sugar chains contain N-acetylneuraminic acid and can be separated by paper electrophoresis. By combination of sequential exoglycosidase digestion and methylation study, their structures were elucidated as Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6 or 4Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 4 or 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.     

Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1

Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) represses translation initiation by binding to eukaryotic initiation factor 4E (eIF4E). 4E-BP1 also binds to the eIF4E homologous protein (4EHP). We show that eIF4E-binding mutants of 4E-BP1 (Y54A and L59A) fail to form heterodimeric complexes with wild-type 4EHP. In addition, the W95A mutant of 4EHP, similar to a homologous mutation in eIF4E, inhibits its binding to wild-type 4E-BP1. Interestingly, 4EHP over-expression instigates a negative feedback loop that inhibits upstream signaling to 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) whereas the 4E-BP1-binding-deficient mutant of 4EHP(W95A) was unable to trigger this feedback loop. Thus, the interaction of 4EHP with 4E-BP1 is necessary for this observed impaired signaling to 4E-BP1 and S6K1.     

Activation of 4-alpha-glucanotransferase activity of porcine liver glycogen debranching enzyme with cyclodextrins

Glycogen debranching enzyme (GDE) is a single polypeptide chain containing distinct active sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Debranching of phosphorylase limit dextrin from glycogen is carried out by cooperation of the two activities. We examined the effects of cyclodextrins (CDs) on debranching activity of porcine liver GDE using a fluorogenic branched dextrin, Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), as a substrate. B5/84 was hydrolyzed by the hydrolytic action of 4-alpha-glucanotransferase to B5/81 and maltotriose. The fluorogenic product was further hydrolyzed by the amylo-alpha-1,6-glucosidase activity to the debranched product, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G8PA), and glucose. alpha-, beta- and gamma-CDs accelerated the liberation of B5/81 from B5/84, indicating that the 4-alpha-glucanotransferase activity was activated by CDs to remove the maltotriosyl residue from the maltotetraosyl branch. This led to acceleration of B5/84 debranching. The extent of 4-alpha-glucanotransferase activation increased with CD concentration before reaching a constant value. This suggests that there is an activator binding site and that the binding of CDs stimulates 4-alpha-glucanotransferase activity. In the porcine liver, glycogen degradation may be partially stimulated by the binding of a glycogen branch to this activator binding site.     

Glycosphingolipids in insects. Chemical structures of ceramide tetra-, penta-, hexa-, and heptasaccharides from Calliphora vicina pupae (Insecta: Diptera)

Four neutal fraction glycosphingolipids, designated components 4-7, were purified from the pupae of Calliphora vicina and isolated by the use of high performance liquid chromatography. Their chemical structures were determined to be: GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; and GlcNAC(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer. By the use of specific exoglycosidases, it was possible to assign anomeric configurations to all the sugar residues present. Analysis of the ceramide moiety by electron-impact mass spectrometry revealed the dominant fatty acid and sphingoid to be arachidic acid (C20:0) and tetradecasphing-4-enine, respectively.     

Tissue Distribution, Genomic Structure, and Chromosome Mapping of Mouse and Human Eukaryotic Initiation Factor 4E-Binding Proteins 1 and 2

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91.5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans ∼16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescencein situhybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21–q22, respectively.     

New pregnane glycosides from the roots of Cynanchum otophyllum

Six new pregnane glycosides with an acyl at C-12 and a straight sugar chain at C-3, namely otophyllosides H-M (1-6), were isolated from the roots of Cynanchum otophyllum (Asclepiadaceae) collected from Eryuan County in Yunnan province of China. Their structures were characterized to be qingyangshengenin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-glucopyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-d-digitoxopyranoside (1), qingyangshengenin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-digitoxopyranoside (2), qingyangshengenin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-digitoxopyranoside (3), qingyangshengenin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-thevetopyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-digitoxopyranoside (4), caudatin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-glucopyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-d-cymaropyranoside (5), caudatin 3-O-beta-d-glucopyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-d-cymaropyranosyl-(1-->4)-beta-d-cymaropyranoside (6), respectively, on the basis of detailed spectroscopic analysis and chemical method.     

Role of CD4 hinge region in GP120 utilization by immunoglobulin domain 1

Immunoglobulin-like domain 1 of CD4 (D1-CD4) promotes HIV infection by binding the envelope glycoprotein (ENV) and exposing its coreceptor-binding site. To study CD4-ENV-coreceptor interactions, we characterized hybrid receptors having domains 1 and 2 of CD4 (D1D2-CD4) joined to the N-terminus of chemokine receptors CCR5, CXCR4, CXCR2, and DARC. Hybrid receptors showed conserved ENV-coreceptor specificity in cell-cell fusion assays. Although D1D2-CD4-CCR5 was sufficient to permit ENV-mediated fusion, D1-CD4-CCR5 and human D1/mouse D2-CD4-CCR5 lacked CD4 function and binding to a neutralizing antibody mapped to D1-CD4. Chimeric D1D2-CD4 joined to CCR5 revealed that the C-terminal 20 residues of human D2-CD4 are required for efficient ENV-mediated fusion. Mutagenesis of hybrid receptors showed the importance of residues forming D1-D2 CD4 interdomain contacts and hinge region proximal residues. Mutagenesis of WT human CD4 confirmed that residues forming D1-D2 interdomain contacts and hinge-region proximal residues contribute positively to CD4 activity in the full-length receptor.     

Sulfated oligosaccharides isolated from the deamination products of heparins

Porcine heparin, whale heparin, and a solvolyzed porcine heparin were deaminated, and sulfated oligosaccharides, compounds 3f, 4f, 3s, 4s, 5, 6, 7s, 10, 11f, 11s, and 13 were isolated from the deamination products by Dowex 1 x 2 (Cl- form) column chromatography and high voltage paper electrophoresis and/or gel filtration on Sephadex G-25. Based on the results of chemical, 1H and 13C NMR spectral analyses, and of Smith degradation, together with previous observations, the structures of these sulfated oligosaccharides are proposed to be as follows: compound 3f, IdUA(2S)alpha 1 leads to GlcNAc alpha 1 leads to 4GlcUA; compound 4f, IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA; compound 3s, IdUA(2S) alpha 1 leads to 4GlcNAc alpha 1 leads to 4 GlcUA beta 1 leads to 4a Man; compound 4s, IdUA alpha 1 leads to 4Glc NAc(6S) alpha 1 leads to 4 GlcUA beta 1 leads to 4aMan; compound 5, IdUA(2S) alpha 1 leads to 4aMan; compound 6, GlcUA beta 1 leads to aMan(6S); compound 7s, IdUA alpha 1 leads to 4aMan(6S); compound 10, IdUA(2S)alpha 1 leads 4GlcNAc(6S)alpha 1 leads to 4 GlcUA beta 1 leads to 4aMan; compound 11f, IdUA(2S) alpha 1 leads 4GlcNAc alpha 1 leads to 4GlcUA beta 1 leads to 4a Man (6S); compound 11s, IdUA alpha 1 leads to GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4aMan(6S); compound 13, IdUA(2S) alpha 1 leads to 4aMan(6S). For ths sulfated disaccharides, the same results as those reported in our previous papers were obtained. On the other hand, the proportion of total sulfated tri- and tetrasaccharides from whale heparin was 1.9 times higher than that from porcine heparine, reflecting a higher content of GlcNAc in the former. Also, the yields of compound 11s from these two heparins were comparable to their anticoagulant activities. In addition, certain 2-O-sulfates on IdUA flanked with GlcNS(6X) (X=H or S) in the heparin molecule are suggested to be important for the activity.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.     

4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function

4-1BB (CD137) is a costimulatory member of the TNFR family expressed on activated T cells. Its ligand, 4-1BBL, is expressed on activated APC. In the mouse, CD8 T cells are preferentially activated by agonistic anti-murine 4-1BB Abs. However, murine 4-1BBL can stimulate both CD4 and CD8 T cells. To date, there are only limited data on the effects of 4-1BBL on human T cell responses. To further understand the role of 4-1BBL in human T cell responses, we compared human CD4 and CD8 T cell responses to transfected human 4-1BBL plus TCR-mediated stimulation. Both human CD4 and CD8 T cells responded to 4-1BBL. The presence of 4-1BBL on the APC led to increased expansion, cytokine production, and the development of cytolytic effector function by human T cells. In unfractionated T cell cultures, CD4 and CD8 T cells could expand to a similar extent in response to signals through the TCR and 4-1BB, as measured by CFSE labeling and by quantitating T cell numbers in the cultures. In contrast to the results with total T cells, isolated CD8 T cells produced less IL-2 and expanded to a lesser extent than isolated CD4 T cells responding to 4-1BBL. Thus, 4-1BBL is most effective when both CD4 and CD8 T cells are included in the cultures. CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells, and CTLA-Ig partially blocked 4-1BBL-dependent IL-2 production. However, a portion of the 4-1BBL-mediated effects were independent of CD28-B7 interaction.     

Electrophysiological properties of heteromeric TRPV4-C1 channels

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4-C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4-C1 and homomeric TRPV4 channels. For heteromeric TRPV4-C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4-C1 channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4-C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.     

Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.     

Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels

With-no-Lysine kinase 4 (WNK4) inhibited ROMK (Kir1.1) channels and the inhibitory effect of WNK4 was abolished by serum-glucocorticoid-induced kinase 1 (SGK1) but restored by c-Src. The aim of the present study is to explore the mechanism by which Src-family tyrosine kinase (SFK) modulates the effect of SGK1 on WNK4 and to test the role of SFK-WNK4-SGK1 interaction in regulating ROMK channels in the kidney. Immunoprecipitation demonstrated that protein phosphatase 1 (PP1) binds to WNK4 at amino acid (aa) residues 695-699 (PP1(#1)) and at aa 1211-1215 (PP1(#2)). WNK4(-PP1#1) and WNK4(-PP1#2), in which the PP1(#1) or PP1(#2) binding site was deleted or mutated, inhibited ROMK channels as potently as WNK4. However, c-Src restored the inhibitory effect of WNK4 but not WNK4(-PP1#1) on ROMK channels in the presence of SGK1. Moreover, expression of c-Src inhibited SGK1-induced phosphorylation of WNK4 but not WNK4(-PP1#1) at serine residue 1196 (Ser(1196)). In contrast, coexpression of c-Src restored the inhibitory effect of WNK4(-PP1#2) on ROMK in the presence of SGK1 and diminished SGK1-induced WNK4 phosphorylation at Ser(1196) in cells transfected with WNK4(-PP1#2). This suggests the possibility that c-Src regulates the interaction between WNK4 and SGK1 through activating PP1 binding to aa 695-9 thereby decreasing WNK4 phosphorylation and restoring the inhibitory effect of WNK4. This mechanism plays a role in suppressing ROMK channel activity during the volume depletion because inhibition of SFK or serine/threonine phosphatases increases ROMK channel activity in the cortical collecting duct of rats on a low-Na diet. We conclude that regulation of phosphatase activity by SFK plays a role in determining the effect of aldosterone on ROMK channels and on renal K secretion.     

Proton-nuclear magnetic resonance study of peracetylated derivatives of ten oligosaccharides isolated from human milk

Proton-nuclear magnetic resonance (NMR) spectra of peracetylated derivatives of ten structurally related oligosaccharides isolated from human milk were measured for solutions in CDCl3 at 360 MHz. The following oligosaccharides were investigated: Gal beta 1 leads to 4Glc-ol (1), GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (2), Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (3), Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (4), Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha) beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (5), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (6), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha)beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (7), Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (8), and a 1:3 mixture of Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol (9) and Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (10). Owing to the strong downfield shifts of the resonances of protons linked to acetoxylated carbons, the problems of signal overlap are less severe and the spin systems of all constituent sugar residues can be assigned fully. The sites of glycosidic linkage can be recognized by the high-field position of the signals of protons linked to those sites; for example, type 1 (Gal beta 1 leads to 3GlcNAc) and type 2(Gal beta 1 leads to 4GlcNAc) saccharide chains can be distinguished. The sequence can be established by observing a nuclear Overhauser effect involving the anomomeric and the aglyconic proton.     

Probing the C4-binding site on C1s with monoclonal antibodies. Evidence for a C4/C4b-binding site on the gamma-domain

The catalytic site for C4 of C1s has been presumed to consist of a C4-binding domain and a proteolytic domain. A mAb to C1s, M81, blocked C4 activation and C4 binding to C1s. M81 recognized the H chain of C1s. Using M81 as a probe, we tried to define C4-binding site on C1s. Plasmin digestion of C1s generated four products of Mr 58,000 (P1), 48,000 (P2), 37,000 (P3), and 27,000 (P4). These products, except for P2, all possessed a 26,000-Da H chain fragment (26k-HF) connected to variable-sized L chain pieces. 26k-HF alone had an ability to interact with M81. Amino-terminal amino acid analysis of 26k-HF mapped the epitope for M81 to domain IV and/or V of gamma-domain of C1s. The gamma-domain therefore contains the C4-binding site. The confirm and further elucidate the role of the C4-binding site for C4, we used a substrate-blotting technique in which labeled C4 was incubated with nitrocellulose membrane-fixed C1s and its fragments. C4 was successfully blotted onto C1s and P1, but not P2-P4; i.e., further degradation of the L chain led to the loss of C4-binding. During the incubation, most of the added C4 was converted to C4b. The binding was augmented, if the proteolytic activity of C1s and P1 was blocked, so that the added C4 remained intact. Although C4b also bound to C1s and P1, its binding was less effective and abolished by the addition of cold C4. Based on these results, the gamma-domain and the L chain constitute the catalytic site of C1s to activate C4 to C4b. Moreover, the generated C4b, although it still has weak affinity for C1s, can be replaced by newly coming C4.     

4-1BB costimulation promotes human T cell adhesion to fibronectin

CD28 and 4-1BB (CD137) are costimulatory molecules for T cells. In this study we investigated the role of 4-1BB in T cell adhesion to fibronectin (FN). Unlike CD28, 4-1BB is present in only a small subset of T cells prepared from fresh human peripheral blood mononuclear cells, but was induced after prolonged TCR/CD28 activation in vitro. 4-1BB-expressing T cells were characteristically unique in their strong responsiveness to FN. Anti-4-1BB cross-linking synergized CD28 costimulation by lowering the threshold of CD3 signal required for CD28-mediated maximal proliferative response. In addition to increasing proliferative responses, 4-1BB promoted T cell adhesion to FN in the presence of CD28 costimulation. 4-1BB-mediated cell adhesion to FN was blocked by anti-beta1 integrin, suggesting that 4-1BB mediates beta1 integrin activation. The role of 4-1BB in inducing CD4(+) T cell adhesion to FN was confirmed by showing that the human leukemic CD4(+) T cell line, Jurkat, when transfected with cDNA encoding 4-1BB, became adherent to FN with anti-4-1BB stimulation. Taken together, our results suggest that 4-1BB-promoted T cell adhesion to extracellular matrix proteins is an important postactivation process for T cell migration.