Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z approximately 13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Gal beta1-4GlcNAc)(n) units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis(x) and sialyl-Lewis(x) determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.

Protozoan parasites of the genus Leishmania produce the novel surface glycoconjugate, lipophosphoglycan (LPG), which is required for parasite infectivity. In this study we show that LPG structure is modified during the differentiation of L. major promastigotes from a less infectious form in logarithmic growth phase to a highly infectious 'metacyclic' form during stationary growth phase. In both stages, the LPGs comprise linear chains of phosphorylated oligosaccharide repeat units which are anchored to the membrane via a glycosyl-phosphatidylinositol glycolipid anchor. During metacyclogenesis there is (i) an approximate doubling in the average number of repeat units per molecule from 14 to 30, (ii) a pronounced decrease in the relative abundance of repeat units with side chains of beta Gal or Gal beta 1-3Gal beta 1-, and a corresponding increase in repeat units with either no side chains or with side chains of Arap alpha 1-2 Gal beta 1- and (iii) a decrease in the frequency with which the glycolipid anchor is substituted with a single glucose alpha 1-phosphate residue. While the majority of the LPG phosphoglycan chains are capped with the neutral disaccharide, Man alpha 1-2Man, a significant minority of the chains appeared to terminate in non-phosphorylated repeat units and may represent incompletely capped species. We suggest that the developmental modification of LPG may be important in modulating the binding of promastigotes to receptors in the sandfly midgut and on human macrophages and in increasing the resistance of metacyclic promastigotes to complement-mediated lysis.

It has been postulated that chromatin modifications can persist through mitosis and meiosis, thereby securing memory of transcriptional states. Whether these chromatin marks can self-propagate in progeny independently of relevant trans-acting factors is an important question in phenomena related to epigenesis. "Adaptive cellular memory" displayed by yeast cells offers a convenient system to address this question. The yeast GAL genes are slowly activated by Gal4 when cells are first exposed to galactose, but their progeny, grown in glucose media, exhibit a fast activation mode upon re-exposure to this sugar. This "galactose memory" persists for several generations and was recently proposed to involve chromatin modifications and perinuclear topology of the GAL genes cluster. Here, we perform a heterokaryon assay demonstrating that this memory does not have a chromatin basis but is maintained by cytoplasmic factor(s) produced upon previous galactose induction. We show that Gal3, the cytoplasmic rate-limiting factor that releases the Gal4 activator, is dispensable for preserving galactose memory. Instead, the important memory determinant is a close Gal3 homolog, the highly expressed Gal1 galactokinase, the residual activity of which preserves memory in progeny cells by rapidly turning on the Gal4 activator upon cells' re-exposure to galactose.

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.

The neuroepithelial layer of the developing eyecup contains multipotential precursor cells that give rise to all of the neurons and the one glial cell type present in the adult retina. Patterning within the retinal neuroepithelium is regulated by cell intrinsic as well as cell extrinsic mechanisms. Although the identity of some of the signaling molecules that regulate retinal development is known, the function of many others, especially members of the Wnt family, has yet to be characterized in the context of retinal development. We undertook a comprehensive in situ hybridization analysis to examine the expression of Wnt pathway components in the developing and adult mouse neural retina. Our findings confirm and extend previous expression studies in mice and other vertebrates, as we show that Wnt-3, -5a, -5b, and -7b are expressed in the neural retina and that there is a dynamic pattern of Wnt receptor (Mouse frizzled [Mfz]) and Wnt antagonist (Secreted-frizzled-related protein [Sfrp]) gene expression in the embryonic and perinatal neural retina. Moreover, we show that Wnt-13 is expressed in the pigment epithelium overlying the distal part of the eyecup and the ciliary margin and that Mfz-4, -6, and -7 are expressed in different regions within the ciliary margin. To determine where activation of canonical Wnt signaling is occurring in the retina, we examined reporter gene expression in TCF/Lef-LacZ mice and we demonstrate that the highest levels of beta-gal activity are found in the ciliary margin, adjacent to and within the Wnt-13 expression domain, implicating Wnt-13 signaling in the development of the ciliary margin and its derivatives.

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.

Gene regulation networks contain recurring circuit patterns called network motifs. One of the most common network motif is the incoherent type 1 feed-forward loop (I1-FFL), in which an activator controls both gene and repressor of that gene. This motif was shown to act as a pulse generator and response accelerator of gene expression. Here we consider an additional function of this motif: the I1-FFL can generate a non-monotonic dependence of gene expression on the input signal. Here, we study this experimentally in the galactose system of Escherichia coli, which is regulated by an I1-FFL. The promoter activity of two of the gal operons, galETK and galP, peaks at intermediate levels of the signal cAMP. We find that mutants in which the I1-FFL is disrupted lose this non-monotonic behavior, and instead display monotonic input functions. Theoretical analysis suggests that non-monotonic input functions can be achieved for a wide range of parameters by the I1-FFL. The models also suggest regimes where a monotonic input-function can occur, as observed in the mglBAC operon regulated by the same I1-FFL. The present study thus experimentally demonstrates how upstream circuitry can affect gene input functions and how an I1-FFL functions within its natural context in the cell.

RNA levels are determined by the rates of both transcription and decay, and a mechanistic understanding of the complex networks regulating gene expression requires methods that allow dynamic measurements of transcription and decay in living cells with minimal perturbation. Here, we describe a metabolic pulse-chase labeling protocol using 4-thiouracil combined with large-scale RNA sequencing to determine decay rates of all mRNAs in Saccharomyces cerevisiae. Profiling in various growth and stress conditions reveals that mRNA turnover is highly regulated both for specific groups of transcripts and at the system-wide level. For example, acute glucose starvation induces global mRNA stabilization but increases the degradation of all 132 detected ribosomal protein mRNAs. This effect is transient and can be mimicked by inhibiting the target-of-rapamycin kinase. Half-lives of mRNAs critical for galactose (GAL) metabolism are also highly sensitive to changes in carbon source. The fast reduction of GAL transcripts in glucose requires their dramatically enhanced turnover, highlighting the importance of mRNA decay in the control of gene expression. The approach described here provides a general platform for the global analysis of mRNA turnover and transcription and can be applied to dissect gene expression programs in a wide range of organisms and conditions.

Cyclic AMP (cAMP) and its receptor protein (CRP) have a dual role in the regulation of the two promoters that control the galactose (gal) operon of Escherichia coli. One promoter, P1, requires cAMP-CRP for activity; the other, P2, is inhibited by these factors. We have examined the interactions site of cAMP-CRP on gal DNA by using two types of protection experiments, involving DNase digestion and methylation by dimethyl sulfate. Our results indicate that cAMP-CRP binds to gal DNA in a segment located between 50 and 24 base pairs preceding the P1 start point for transcription. Although the location of the cAMP-CRP interaction site is clearly different in gal and lac DNA, comparison of the DNA sequences suggests a similar recognition sequence. The location of the cAMP . CRP-binding site in gal further suggests that protein-protein interactions between RNA polymerase and cAMP . CRP play an important role in transcription initiation at the gal and possibly other cAMP-dependent promoters.

Uropathogenic Escherichia coli frequently express P-pilus adhesins that recognize Gal alpha (1-4)Gal-containing glycoconjugates. The P-pilus adhesin of the E. coli isolate J96 is encoded by the pap gene cluster and has been shown to agglutinate P1-erythrocytes. We now describe a novel gene cluster from J96, prs, which is responsible for the agglutination of sheep erythrocytes. The structurally related gene clusters both expressed pili exhibiting the F13 antigen. Analysis of mutants of cloned prs sequences, together with trans-complementation of pap and prs genes, identified the sheep-specific adhesin as the 37-kD PrsG protein. The prsG gene occupies the equivalent position in prs as occupied by papG, which specifies the Gal alpha (1-4)Gal-specific adhesin of pap. PrsG was shown to be structurally distinct from PapG since PapG-specific antiserum did not cross-react with PrsG. Using a solid phase glycolipid receptor binding assay, PrsG was found to specify preferential binding to the Forssman antigen, a major constituent of sheep erythrocyte membranes. The binding epitope was identified as the GaINAc alpha (1-3)GaINAc moiety. This is the first direct evidence that serologically identical pili may present antigenically distinct adhesins, each capable of binding to a specific receptor.

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining demonstrated expression of beta-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.

An evaluation has been made of the E. coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed.

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho's ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H(2)O(2)-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H(2)O(2)- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated beta-gal staining showed that Klotho protein interferes with H(2)O(2)-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H(2)O(2)-induced apoptosis and cellular senescence in vascular cells.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

BACKGROUND

Fabry disease is an X-linked disorder of glycosphingolipid catabolism that is the result of an intracellular deficiency in the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). This enzymatic defect results in the accumulation of globotriaosylceramide (Gb(3)) within cells and causes progressive neurological, cardiovascular and renal dysfunction. Our objective is to describe the safety and renal effects of long-term enzyme replacement therapy.

METHODS

This was a single centre, prospective open-label treatment trial in 25 adult male Fabry patients who had completed a 6-month randomized placebo-controlled study and subsequently enrolled in an open-label extension study. Patients were treated every other week with agalsidase alfa (0.2 mg/kg) infused intravenously over 40 min. The main outcome measures were safety, antibody response and renal glomerular filtration rate (GFR).

RESULTS

During the 4-4.5 years of enzyme replacement therapy, all eligible subjects were able to transition to home therapy. Eight patients developed persistent IgG antibodies to agalsidase alfa, but IgE antibodies were not detected in any patient. The development of IgG antibodies appeared not to affect any clinical end points. Estimated GFR remained stable in subgroups of patients with Stage I (GFR >90 ml/min) or Stage II (GFR 60-89 ml/min) chronic kidney disease at baseline. In contrast, in the subgroup of patients with Stage III chronic kidney disease (GFR 30-59 ml/min), the slope of the decline in GFR was reduced compared with comparable historical controls, suggesting that enzyme replacement therapy was slowing the decline of renal function in this susceptible population.

CONCLUSIONS

Long-term enzyme replacement therapy with agalsidase alfa is safe and may slow the progressive decline in renal function that was commonly observed in adult males with Fabry disease.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Heparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcA-Gal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N-deacetylation and N-sulfation of glucosamine, C5-epimerization of GlcA and multiple O-sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate.

The coordinated activity of Runx2 and BMP/TGFbeta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGFbeta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391. Thus the Smad interacting domain (SMID) of Runx2 (432-391) is embedded in the well-defined nuclear matrix targeting signal (NMTS) that mediates intranuclear trafficking. The SMID suffices as an interacting module when fused to the heterologous Gal-4 protein. Formation of the Runx2 and Smad complex is dependent on Runx2 phosphorylation through the MAPK signaling pathway, as determined by co-immunoprecipitation studies. We established that all SMID/NMTS deficient Runx2 mutants do not show in situ association with Smad in the nucleus nor do they support BMP2-mediated osteogenic induction of the mesenchymal C2C12 cell line. Thus, we provide direct evidence that the SMID/NMTS domain (391-432) of Runx2 is essential for BMP2-mediated osteoblast differentiation. Our findings suggest that TGFbeta/ BMP2 signaling, MAPK dependent phosphorylation, and Runx2 subnuclear targeting converge to induce the osteogenic phenotype.

To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.

The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.

The 21 kd NusG protein is essential for E. coli viability. Cells depleted for NusG were defective for factor-dependent transcription termination. Rho-induced polarity in the gal operon and the Rho-dependent lambda tR1 and lambda tL1 terminators were suppressed in NusG-deficient cells. NusG depletion inactivated the phage HK022 Nun termination factor. In contrast, the factor-independent lambda tl terminator was fully active in NusG-depleted cells and could be suppressed by phage lambda N function.

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.

Galectin-3 (Gal-3), a member of the beta-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser(6) by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser(6) regulates Gal-3 function. We have generated serine-to-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser(6) phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3.