Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2

The human low affinity FcγRII family includes both the activating receptor FcγRIIA and the inhibitory receptor FcγRIIB2. These receptors have opposing signaling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. We demonstrate that upon engagement by multivalent aggregated human IgG, FcγRIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while FcγRIIB2 dissociates from the ligand and is routed separately into the recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase activity or ubiquitylation of receptor lysine residues. The sorting of FcγRIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of FcγRIIA and FcγRIIB2, the receptors are sorted independently to distinct final fates after dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behavior of different Fcγ receptors.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

The regulatory mechanism of extracellular Hsp90{alpha} on matrix metalloproteinase-2 processing and tumor angiogenesis

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone that is essential for eukaryotic homeostasis. Hsp90α can also be secreted extracellularly, where it has been shown to be involved in tumor metastasis. Extracellular Hsp90α interacts with and promotes the proteolytic activity of matrix metalloproteinase-2 (MMP-2). However, the regulatory mechanism of Hsp90α on MMP-2 activity is still unknown. Here we show that Hsp90α stabilizes MMP-2 and protects it from degradation in tumor cells. Further investigation reveals that this stabilization effect is isoform-specific, ATP-independent, and mediated by the interaction between the Hsp90α middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that secrete more Hsp90α in their proliferating status. Furthermore, endothelial cell transmigration, Matrigel plug, and tumor angiogenesis assays demonstrate that extracellular Hsp90α promotes angiogenesis in an MMP-2-dependent manner. In sum, this study provides new insights into the molecular mechanism of how Hsp90α regulates its extracellular client proteins and also reveals for the first time the function of extracellular Hsp90α in promoting tumor angiogenesis.     

The Neurospora Peptide:N-Glycanase Ortholog PNG1 Is Essential for Cell Polarity despite Its Lack of Enzymatic Activity

Secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. The cytoplasmic peptide:N-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 S proteasome. In contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:N-glycanase activity remains unclear. Here we show that the PNG1 ortholog from the filamentous ascomycete Neurospora crassa is an essential protein, and its deletion results in strong polarity defects. PNG1 and its predicted binding partner RAD23 have distinct functions in N. crassa and are involved in cell wall integrity and DNA repair, respectively. Moreover, wild type PNG1 has substitutions in essential catalytic amino acids, and its deglycosylation activity is lost. These substitutions are conserved in many PNG1 orthologs of the fungal kingdom, implying a so far unrecognized enzyme-independent function of PNG1 that may only become apparent in highly polar cells such as fungal hyphae.     

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5′-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serine- and arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.