Johannes Müllegger

Hyal2-less active, but more versatile?

Hyal2 is one of several hyaluronidases present in vertebrates. The human gene encoding this enzyme is present on chromosome 3p.21.3, close to two additional hyaluronidase genes. cDNAs encoding Hyal2 homologues have been characterized from mouse and Xenopus laevis. These enzymes hydrolyze high molecular mass hyaluronan to intermediates of approximately 20 kDa, a finding which implies that structural domains of this size exist in this polysaccharide which was mostly thought to be a random coil. Hyal2 enzymes have an acidic pH-optimum with an activity that is considerably lower than observed for other types of hyaluronidases. Originally considered to be a typical lysosomal enzyme, more recent evidence has shown that Hyal2 proteins can also be exposed on the cell surface bound to the plasma membrane via a GPI anchor. Hyal2 is present in many tissues, one exception being the adult brain. In this tissue, the gene is silenced after birth by methylation. Current evidence about the role of Hyal2 in tumor growth, inflammation and frog embryogenesis is discussed.

Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis

The localization of hyaluronan has been determined in tailbud stage embryos of Xenopus laevis using a neurocan-alkaline phosphatase fusion protein. This polysaccharide was located between the germ layers and enriched in mesenchyme, the lumen of the neural tube, the embryonic gut, the hepatic cavity and the heart. A full-length cDNA for a hyaluronan synthase, Xhas2 has been cloned. The expression pattern of Xhas1 and 2 is closely similar to the distribution of hyaluronan in the embryo. Xhas1 produces hyaluronan with a molecular mass of around 40-200 kDa, while the product formed by Xhas2 has a molecular mass above 1 million Da.

Thioglycosynthases: double mutant glycosidases that serve as scaffolds for thioglycoside synthesis

A double mutant, retaining glycosidase that lacks both the catalytic nucleophile and the catalytic acid/base residues efficiently catalyzes thioglycoside formation from a glycosyl fluoride donor and thiosugar acceptors.

Thermostable Glycosynthases and Thioglycoligases Derived from Thermotoga maritima β-Glucuronidase

Uronic acid-containing glycoconjugates are found in a number of different and important contexts. Examples include pectins and hemicelluloses within plant cell walls, glycosaminoglycans (GAGs) within the mammalian extracellular matrix, capsular poly ACHTUNGTRENNUNGsaccharides of bacteria and glucuronide conjugates formed as a means of solubilisation and clearance of unwanted molecules. Of these, GAGs such as heparin, heparan sulfate, chondroitin sulfate and hyaluronan are of particular importance in a variety of biological processes, and analogues of these structures might well be useful as therapeutic agents. 2] While the chemical syntheses of short oligosaccharide fragments of GAGs have been achieved, these syntheses are surprisingly challenging, and their scale-up to the levels needed for clinical trials remains challenging. An alternative synthetic approach involves the use of enzymes, and, in that regard, ACHTUNGTRENNUNGadvances have been made in several areas. The enzymes ACHTUNGTRENNUNGinvolved in GAG biosynthesis, the glycosyltransferases, have proved problematic, largely because they are generally closely membrane-associated. However, considerable success has been achieved by DeAngelis’ group with hyaluronan synthase, and small-scale syntheses with recombinant enzymes are now available. 5] Glycosidases run “in reverse” provide the other approach, and, indeed, Kobayashi et al. have successfully assembled hyaluronan and chondroitin sulfate oligosaccharides from disaccharide precursors, converted into their oxazolines, by use of endo-hexosaminidases. 7] An alternative strategy for oligosaccharide assembly involves the use of retaining glycosidases in which either the catalytic nucleophile (glycosynthases) or the acid/base catalyst (thioACHTUNGTRENNUNGglycoligases) is mutated (Scheme 1). Glycosynthases are hydroACHTUNGTRENNUNGlytically incompetent mutants that can, nevertheless, effect efficient glycosyl transfer from a glycosyl fluoride sugar donor of opposite anomeric configuration to that of the natural substrate. The glycosyl fluoride donor binds and acts as a mimic of the normal glycosyl enzyme. A range of such enzymes has now been produced, and directed evolution has generated highly efficient catalysts (kcat 90 s ). Thioglycoligases carry out glycosyl transfer from an activated glycosyl donor of normal configuration to a thiosugar acceptor, with formation of a thioglycosidic linkage. In this case, the activated leaving group “complements” the absence of the acid catalyst in the formation of the glycosyl enzyme, while the highly nucleophilic thiol ACHTUNGTRENNUNG(ate) “complements” the missing general base catalyst. While a range of glycosynthases and thioglycoligases has now been produced, and directed evolution approaches have been employed to boost rates and alter specificities, there have been no reports to date of either glycosynthases or thioglycoligases that transfer glycuronyl residues. Given the importance of these structures and the difficulties noted earlier with effective synthesis, we investigated the potential for both classes of mutant enzymes in the synthesis of glycuronyl linkages. An appropriate candidate glycuronidase for the generation of both a glycosynthase and a thioglycoligase was the thermostable b-glucuronidase of Thermotoga maritima (TMGUA), a member of GH family 2 (http://afmb.cnrs-mrs.fr/ CAZY/), particularly as the identities of both the nucleophile (E476) and the acid/base catalyst (E383) have recently been confirmed.

Thioglycoligase-Based Assembly of Thiodisaccharides: Screening as β-Galactosidase Inhibitors

Carbohydrates in cells play very important roles in a wide range of biological processes, and impact health and disease.[1] Consequently, interference with the recognition and processing of carbohydrates is a strategy for drug development that is gaining favour. Examples of drugs that are already in the clinic include amylase inhibitors for the reduction of blood glucose levels and sialidase inhibitors as anti-influenza drugs.[2] A newer class of molecules currently under development are those that stabilise otherwise unstable mutant forms of lysosomal glycosidases, and thereby chaperone them to their lysosomal location and bypass proteasomal degradation. These pharmacological chaperones are typically inhibitors, but can be used to rescue deficient glycosidase activity in lysosomes and thereby provide a potential treatment for this class of storage disorders.[3] The glycosidase inhibitors employed in such approaches are typically high affinity transition-state analogues, but such compounds often do not have high linkage specificity for the specific target glycosidase since they typically do not contain components of the “aglycone”. Further, very high affinities are not desirable in this application since potent inhibitors would not be released upon reaching the lysosome. Alternatively, noncleavable substrate analogues can be used as competitive inhibitors for both glycosidases and carbohydrate-binding proteins. Although typically less potent, such reagents are generally more specific. Some of the best such analogues are thioglycosides, wherein a sulfur atom replaces the intersugar glycosidic oxygen of the normal substrate. These are generally good mimics of the natural substrate, but are recalcitrant to cleavage by essentially all glycosidases.[4]

Degradation of hyaluronan by a Hyal2-type hyaluronidase affects pattern formation of vitelline vessels during embryogenesis of Xenopus laevis

A Hyal2-type hyaluronidase of Xenopus laevis (Xhyal2) was characterized by molecular cloning, biochemical analysis and ectopic overexpression in embryos. When expressed in Xenopus oocytes, Xhyal2 exists as a soluble protein in the extracellular space and in intercellular compartments as well as being attached to the cell surface through a glycosyl-phosphatidyl-inositol anchor. This enzyme specifically degrades hyaluronan not only at acidic pH values but more slowly also under physiological conditions. Xhyal2 is differentially expressed during embryogenesis. Particularly striking is the high level of expression in the developing brain, the head mesenchyme and the pronephros. Elevated levels of mRNA were also found in endothelial cells which will later form vascular structures. Ectopic overexpression of Xhyal2 in frog embryos causes loss of hyaluronan in the cellular environment. This causes severe defects in the assembly of the highly structured plexus of the vitelline vessels from prevascular endothelial cells. Our data support the notion that the level of Xhyal2 expression determines the organization of the extracellular environment so that cells can merge and/or migrate within an originally impenetrable matrix.

'Piggy-back' transport of Xenopus hyaluronan synthase (XHAS1) via the secretory pathway to the plasma membrane.

Abstract Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.

Identification of the catalytic nucleophile in Family 42 β‐galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis

The mechanism‐based inhibitor 2,4‐dinitrophenyl 2‐deoxy‐2‐fluoro‐β‐d‐galactopyranoside (DNP2FGal) was used to inactivate the Family 42 β‐galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl‐enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high‐performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu‐295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.

Xenopus brevican is expressed in the notochord and the brain during early embryogenesis

A complete cDNA encoding the Xenopus laevis homologue of the aggrecan/versican family member, brevican (Xbcan) was cloned from an embryonic stage 42 cDNA library. In the deduced amino acid sequence, 1152 in length, similarity to the hyaluronan-binding (link) domains of brevicans from other species were present in the N-terminal region as well as EGF-, lectin- and complement regulatory protein-like domains in the C-terminal part, the latter three being characteristic for brevican found within the extracellular matrix (J. Biol. Chem. 269 (1994) 10119). Indeed, Xbcan was secreted into the extracellular space as a soluble protein when expressed in oocytes. No cDNAs encoding a GPI-anchored bcan variant could be isolated from that cDNA library. During embryonic development, the expression of this gene was first observed in the notochord of neurula stage embryos. In addition to this, in tailbuds, Xbcan was also found to be expressed within the fifth and sixth rhombomere of the hindbrain. In tadpole stage embryos, expression was furthermore observed in periventricular regions of the developing brain and the rostral part of the spinal cord.

Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH1

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH‐20‐like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore‐assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane‐bound hyaluronan‐degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.