Day 3 :
Keynote Forum
Konrad Sandhoff
LIMES Institut, Germany
Keynote: Membrane lipids regulate glycosphingolipid catabolism, their enzymes and lipid binding proteins
Time : 10:05-10.30
Biography:
Konrad Sandhoff completed his PhD in biochemistry in Munich. After research stays in Munich, Israel and the USA he became a full professor of biochemistry at the University of Bonn in 1979. Since 2007 he is a senior professor at the LIMES institute, Bonn. Major Research Interests: Molecular life sciences: analysis and pathobiochemistry of lysosomal (glyco-)sphingolipid storage diseases, structure and function of lysosomal enzymes and lipid binding proteins, topology of endocytosis and glycolipid metabolism, and regulation of glycolipid biosynthesis. He has published more than 480 peer-reviewed papers. Among many other prizes he also received the International Glycoconjugate Organization Award (2005).
Abstract:
Cholesterol and sphingolipids (SLs) are stabilizing compounds of eukaryotic plasma membranes. Together with phospholipids (PLs) they reach luminal vesicles of the endolysosomal compartment as platforms for membrane degradation. A maturation process removes lipids inhibiting lysosomal catabolism from the luminal vesicles. Sphingomyelin (SM) is hydrolyzed by acid sphingomyelinase, facilitating cholesterol export to the cytosol by NPC2 and NPC1. SM and cholesterol poor luminal vesicles then serve as platforms for glycosphingolipid degradation in the lysosomes employing soluble hydrolases, SAPs (sphingolipid activator proteins) and anionic PLs as stimulators. We reconstituted the catabolic proteins on liposomal surfaces, mimicking luminal vesicles of the lysosomes as platforms for SL degradation. Liposomes without anionic PLs and with no net surface charge generated only negligible and physiologically irrelevant catabolic rates even at lysosomal pH values. Incorporation of anionic PLs into the SL-carrying liposomes, however, stimulated the catabolic rate by up to more than an order of magnitude. We now found, that the incorporation of cholesterol or SM into the SL carrying liposomal membranes generated a strong inhibition of ganglioside GM2 hydrolysis and the transfer of membrane lipids between liposomal vesicles by SAPs, even in the presence of anionic phospholipids. Ongoing in vitro studies indicate that PM-stabilizing lipids, i.e. SM and cholesterol, inhibit several steps of lysosomal SL and glycosphingolipid catabolism and also lipid solubilisation as studied by Surface Plasmon Resonance and intervesicular (glyco-) lipid transfer activities of several SAPs and NPC2, even in the presence of activating anionic phospholipids.
- Track 8: Synthesis and Biological Role of Glycans
Location: Independence C
Chair
Konrad Sandhoff
LIMES Institut, Germany
Co-Chair
Barbara Klajnert-Maculewicz
University of Lodz, Poland
Session Introduction
John F. Robyt
Iowa State University, USA
Title: Recent advances for the mechanisms involved in glycan biosynthesis
Time : 10:30-10.50
Biography:
Prof. John F. Robyt, Short Bio-sketch: John Robyt received a B.S. in chemistry from St. Louis University in 1958. In the summer of 1957, he received an Internship at the USDA Laboratory in Peoria, IL. He then went to Iowa State University in 1958 to study for a Ph.D., joining Prof. Dexter French’s group in Carbohydrate Chemistry and Enzymology, where he received a Ph.D. in 1962, publishing four papers: on the Action Pattern and Mechanism of Bacillus amyloliquefaciens α-amylase, Bacillus polymyxa ß-amylase, porcine pancreatic α-amylase, the separation and large-scale purification of maltodextrins by charcoal column chromatography, and the development of ascending and descending paper chromatography of carbohydrates. He then went to Louisiana State University in Baton Rouge, LA on a Teaching Postdoctoral Fellowship and taught seven courses in chemistry and biochemistry; then the next year (1963) he received a NIH, Research Fellowship, to work and study at the Lister Institute of Preventive Medicine in London, England, under Prof. William J. Whelan, and published 4 papers. In September 1964, he returned to Iowa State University, as an Instructor and Research Associate with Prof. Dexter French. He was appointed an Assistant Professor in 1967, Associate Professor in 1974, and a Professor in 1982 in the Department of Biochemistry, Biophysics, and Molecular Biology. Besides working on Starch Chemistry and Enzymology, he pursued studies on Dextran Chemistry, and Enzymology, and the formation and prevention of dental plaque, supported by the NIH. He has pursued the use of TLC of carbohydrates, use of NMR to study enzyme mechanisms, application of the use of radioisotopes, and analytical methods of carbohydrates and enzymes, such as reducing-value methods, total carbohydrates by phenol sulfuric acid, and several chromatographic methods, such as ascending and descending paper chromatography, TLC, and column chromatographic methods for separating carbohydrates and enzymes.
Abstract:
Glycan biosynthesis has recently undergone a revolutionary change as to the understanding of how both α- and ß-linked glycans are biosynthesized. Back in the 1940’s C. S. Hanes observed that potato phosphorylase transferred 2-3 glucose units from α-D-Glc-1-P to the nonreducing ends of starch chains. This observation set the stage that starch biosynthesis required a primer. In the 1960’s Luis Leloir found that starch was biosynthesized from ADPGlc. In the late 1960’s, Robbins et al. using pulse and chase techniques found that Salmonella O-antigen was biosynthesized by the transfer of a tetra-saccharide from bactoprenol pyrophosphate to the reducing-ends of a growing chain. Four years later Ward and Perkins showed that the bacterial cell wall, murein, was also biosynthesized by the addition of the NAG-NAM-pentaphosphate to the reducing-ends of growing chains. A year later Robyt, et al. using pulse and chase experiments showed that dextran was biosynthesized by the addition of glucose from sucrose to the reducing ends of a growing dextran chains. Some years later (2007) they showed a two-site insertion mechanism for dextran biosynthesis in which glucose is added to the reducing-ends of growing dextran chains. In 2012, Mukerjea & Robyt showed that the de novo biosynthesis of starch chains by potato starch-synthase adds glucose from ADPGlc to the reducing-ends of growing starch chains. Mukerjea, McIntyre, and Robyt also found that Tris-buffers were potent inhibitors of starch-synthase and has been responsible for the perpetuation of the primer myth for starch biosynthesis, as the putative primers partially reverse the Tris-buffer inhibition.
Konrad Sandhoff
LIMES Institut, Germany
Title: Lysosomal & extracellular GlcCer degradation: Protein & lipid modifiers
Time : 10:50-11:10
Biography:
Konrad Sandhoff completed his PhD in biochemistry in Munich. After research stays in Munich, Israel and the USA he became a full professor of biochemistry at the University of Bonn in 1979. Since 2007 he is a senior professor at the LIMES institute, Bonn. Major Research Interests: Molecular life sciences: analysis and pathobiochemistry of lysosomal (glyco-)sphingolipid storage diseases, structure and function of lysosomal enzymes and lipid binding proteins, topology of endocytosis and glycolipid metabolism, and regulation of glycolipid biosynthesis. He has published more than 480 peer-reviewed papers. Among many other prizes he also received the International Glycoconjugate Organization Award (2005).
Abstract:
Lysosomal sphingolipid degradation requires the presence of water-soluble hydrolases, SAPs, anionic phospholipids like BMP, and an acidic pH value. Inherited defects of catabolic hydrolases or SAPs cause various sphingolipidoses. SAPs are membrane-perturbing proteins which facilitate glycolipid digestion by presenting insoluble lipid molecules to soluble catabolic enzymes. SAPs (the GM2-activator and saposins A-D) bind to lipid bilayers and mobilize lipids out of them at acidic pH values. As demonstrated by plasmon resonance studies for saposins A and B, low cholesterol levels and increasing concentrations of BMP favour lipid extraction and membrane disintegration. Variant saposins as identified in patients with Krabbe disease and metachromatic leukodystrophy, respectively, are deficient in mobilizing membrane lipids. The inherited absence of all four saposins (A-D) causes a severe membrane and sphingolipid storage disease, also disrupting the water permeability barrier of the skin. Saposins and glucosylceramidase are also involved in the extracellular catabolism of ultralongchain acylglucosylceramides, key components for the generation of the extracellular lipid layers forming the immune and the water permeability barrier in the stratum corneum of the mammalian skin. Their complete functional deficiency causes perinatal fatal diseases of the collodion baby type. Ongoing in vitro studies indicate that PM-stabilizing lipids, i.e. SM and cholesterol, inhibit several steps of lysosomal SL and glycosphingolipid catabolism, and also lipid solubilisation as studied by Surface Plasmon Resonance and intervesicular (glyco-) lipid transfer activities of several SAPs and NPC2, even in the presence of activating anionic PLs.
Jiahua Jay Xie
North Carolina Central University, USA
Title: Cytoprotective properties of plant-produced asialoerythropoietin (asialo-rhuepo)
Time : 11:25-11:45
Biography:
Jiahua (Jay) Xie has completed his PhD in Biophysics at the age of 31 from Zhejiang University and postdoctoral studies from Departments of Genetics and Horticultural Science, North Carolina State University. He served as a Senior Scientist at the Vector Research Inc. (a subsidiary of Vector Group Ltd) for five years. Currently, he is an Associate Professor in the Department of Pharmaceutical Sciences and the PI of the BRITE, NCCU. He has published more than 50 papers in reputed journals and has been serving as an editorial board member of Journal of Zhejiang University (Agriculture and Life Sciences).
Abstract:
Asialo-rhuEPO, a non-hematopoietic recombinant human erythropoietin (EPO) derivative lacking sialic acid, has been reported to display broad tissue-protective effects against damage triggered by ischemia/reperfusion, hypoxia or cytotoxic agents in the brain, the heart, the kidneys and the liver. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. It is known that plants can synthesize complex N-glycans similar to mammals, but lack sialylating capacity. In our lab, we generated stable transgenic tobacco lines co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes to produce asialo-rhuEPO (asialo-rhuEPOP) lacking sialic acid, but bearing mammalian-type β1,4-galactose residues. We also developed an efficient purification system for isolating asialo-rhuEPOP from leaf tissues both at small scale (grams) and medium scale (kilograms). Purified asialo-rhuEPOP from transgenic tobacco leaves was found to have superior cytoprotective effect than rhuEPOM in protecting neuronal-like mouse neuroblastoma cells, murine HL-1 cardiomyocytes and pancreatic β-cells from staurosporine-induced cell death. These milestone studies have set the stage for the future investigations on its tissue-protective effects and action mechanisms in various animal models of tissue injury.
Guijun Wang
Old Dominion University, USA
Title: Design, synthesis and characterization of glycolipids and glycoclusters
Time : 11:45-12:05
Biography:
Guijun Wang has completed her PhD in 1999 from Michigan State University and postdoctoral research at Yale University in 2002. She started her independent career as an assistant professor at the University of New Orleans in 2002. In 2012, she relocated to Old Dominion University (ODU), currently she is a professor of Chemistry and Biochemistry at ODU. Her research interests include synthetic methodology development; asymmetric synthesis; and the synthesis and study of carbohydrate based self-assembling systems. She has co-authored 50 peer reviewed papers and is the co-inventor on 20 international and US patents.
Abstract:
Glycolipids and glycoclusters that are able to form self-assembled supramolecular structures are interesting compounds with potential applications as new materials for biomedical research. Our group has a long interest in the design, synthesis and study of various monosaccharide derivatives and we have discovered several new classes of glycolipids based low molecular weight gelators (LMWGs). These compounds form unique soft materials such as organogels or hydrogels that may be useful in biomedical research. They can be used as advanced functional materials for controlled release drug delivery and enzyme immobilization. Using naproxen as a model, we have shown that acid sensitive glucosamine derivatives are effective compounds for controlled release of naproxen from the gel matrix. By incorporating photosensitive functional groups, we have also synthesized and characterized polymerizable diacetylene containing organogels. The resulting polydiacetylenes gels are useful as stimuli-responsive soft materials. Besides these systems, several branched molecules with sugar moieties at the periphery have also been designed and synthesized. These compounds have accurate molecular weight and can form interesting molecular assemblies. The dendritic glycolipids have shown enhanced self-assembling tendencies, which mimic the multivalency effect. In this presentation, our recent studies on glycolipids that can form supramolecular gels and self-assembling glycoconjugates will be discussed.
David Ben-Menahem
Ben-Gurion University of the Negev, Israel
Title: O-glycosylation and protein evolution: the case of the LHï¢ to CGï¢ development
Time : 12:05-12:25
Biography:
David Ben-Menahem has completed his PhD at Tel-Aviv University in Tel-Aviv Israel, and did his postdoctorate studies at Washington University Medical School, in St. Louis Missouri, USA. He is at the department of Clinical Biochemistry and Pharmacology at Ben-Gurion University of the Negev in Beer-Sheva, Israel. His major research focus is related to structure-function studies of the gonadotropins which are members of the glycoprotein hormone family.
Abstract:
The glycoprotein hormones LH, FSH and CG are non-covalent heterodimers composed of the common ï¡ and hormone specific ï¢ subunit. The subunits contain N-linked glycans, which are important for the folding, heterodimer assembly and bioactivity of the hormone. In addition, the carboxy-terminal region of the CGï¢ subunit is O-glycosylated, and this unique domain (known as the CTP) extends the circulatory survival of CG relative to the other glycoprotein hormones. While the genes encoding the ï¡, LHï¢ and FSHï¢ subunits are generic to vertebrates, the CGï¢ gene is restricted to primates and equids. This is curious because the CGï¢ gene presumably evolved from the ancestral LHï¢ gene following only a small set of mutations, and the resulting O-glycosylated CTP confers new hormonal properties to CG relative to LH that seems advantageous to maintain early gestation. To address this restricted evolution, we combined bioinformatics, in-vitro and in-vivo experiments that suggest a) the potential of the LHï¢ to CGï¢ transformation is present in several animal phyla, and b) the ability of a CTP domain to have the clustered O-glycans is important for the CGï¢ development. Additional studies with the equine CTP-extended ï¢ subunit suggest that this subunit, which is expressed in both in the pituitary and placenta of equids integrates intracellular properties that diverged in the LHï¢ and CGï¢ subunits of primates that are expressed in different tissues. Our studies demonstrate a potential role for the CTP O-glycosylation in the LHï¢ to CGï¢ evolution, and a link between tissue expression and subunit characteristics.
Shigeomi Horito
Tokyo University of Science, Japan
Title: Reconstruction of a transmembrane protein tetraspanin (CD9) into lipid bilayer by interaction of ganglioside GM3 and tetraspanin
Time : 12:25-12:45
Biography:
Dr. Horito has completed his PhD from Tokyo Institute of Technology in organic synthesis of carbohydrates and postdoctoral studies from Hamburg University of Germany as Humboldt researcher. He is an Associate Professor in the Dept. of Biological Science & Technology at Tokyo University of Science. He has published more than 25 papers in reputed journals.
Abstract:
Tetraspanin is four times transmembrane protein which constructs a super molecular complex with ganglioside GM3 and CD81 to regulate cell proliferation. Cancer proliferation is also regulate this super molecular complex. It is exciting challenge to elucidate the role of ganglioside GM3 in the super molecular complex. Guofei Son et al reported reconstruction of transmembrane protein aquaporine Z into lipid bilayer by interaction of nickel chelate and histidine tag. We tried to reconstruct tetraspanin by interaction of only natural compounds. GM3/dipalmitoylphosphatigylcholine/dioreoilphosphatigylcholine(1/9/9) lipid-monolayer indicate 2 types domains (0.9 nm high and 2.1 nm high). Hetero-bilayer indicates 2 types domains (6.7 nm high and 12.5 nm high), which is given tetraspanin to indicate 3 types domains (6.1 nm high, 11.7 nm high and 15.6 nm). The highest domain indicates the reconstruction of tetraspanin into the lipid-bilayer.
Barbara Klajnert-Maculewicz
University of Lodz, Poland
Title: Sugar-modified PPI dendrimers as carriers of anti-leukemia drugs
Time : 13:30-13:50
Biography:
Barbara Klajnert-Maculewicz has completed her Ph.D in 2002 from the University of Lodz, Poland and postdoctoral studies from the McMaster University, Ontario, Canada. She is a professor at the University of Lodz, Poland and an external scientific member at Leibniz IPF in Dresden, Germany. She is a co-author of 2 books and 9 chapters in monographs. She has published more than 90 papers in reputed journals (h-index 23). In the years 2009-2012 she was the Management Committee Chair of COST Action TD0802 “Dendrimers in biomedical applications” that gathered 24 countries. She has been awarded L’Oréal-UNESCO Fellowship for Women in Science.
Abstract:
Anticancer drugs such as cytarabine (araC) belong to nucleoside analogues (NAs). NAs are commonly used in the treatment of acute myeloid leukemia, acute lymphocytic leukemia, and lymphomas. AraC acts by interfering with newly synthesized nucleic acids or by modifying physiological nucleosides metabolism. Like most nucleoside analogs, araC is administered as an inactive prodrug and requires specialized nucleoside transporters to cross plasma membranes. Inside a cell, araC is activated to cytotoxic 5’-triphosphates form (araCTP) by intracellular kinases. Unfortunately, a therapy based on cytarabine has its limitations due to several primary and acquired resistance mechanisms that arise during prodrug activation steps. It may lead to inefficient concentration of the therapeutics in cancer cells. Carrier systems that would deliver active forms of NAs are currently seeking. It has been demonstrated that polypropylene imine (PPI) dendrimers with a partially modified surface by maltose residues (PPI-m) easily form complexes with negatively charged 5’-triphosphates of nucleoside analogues. It happens due to the presence of protonated primary and tertiary amino groups. PPI-m dendrimers are non-toxic and highly biocompatible. Moreover, PPI-m dendrimers protect bound drug molecules from enzymatic degradation. Complexes of araCTP and PPI-m dendrimers show enhanced cytotoxic activity against an acute myeloid lukemia cell line 1301 in comparison with free cytarabine and 5’-triphosphate of cytarabine. Thus, PPI-m dendrimers improve stability of NAs and efficiently deliver the active drug forms directly to cancer cells. To sum up, maltose-modified polypropylene imine dendrimers are attractive systems as anticancer drug carriers, especially with a vision to apply them when drug-resistance occurs.
Jan Willem Kok
University of Groningen, Netherlands
Title: Glycosphingolipids enhance transfection efficiency in gm95 cells
Time : 13:50-14:10
Biography:
Jan Willem Kok has completed his PhD at the age of 31 from Groningen University. He is associate professor at the Cell Biology Department of the University Medical Center Groningen, University of Groningen in The Netherlands. He has published more than 70 peer-reviewed papers in reputed journals. His research interest includes sphingolipid biology, lipid rafts, and ABC transporters.
Abstract:
We investigated whether glycosphingolipids in the plasma membrane of cells affect transfection efficiency mediated by cationic lipoplexes. The ideal model cell line GM95 was used, which lacks glycosphingolipids due to mutated glucosylceramide synthase. GM95-GCS cells were generated, which stably express active glucosylceramide synthase. These cells display enhanced transfection efficiency of the reporter gene GFP and enhanced binding/uptake of lipoplexes compared to mock-transfected GM95 cells. The latter effect was mimicked by loading mock-transfected GM95 cells with GM3, but not in case of GM1. We conclude that glycosphingolipids enhance transfection efficiency of cationic lipoplexes and this likely is the result of increased binding/uptake of lipoplexes mediated selectively by GM3.
Miki Hara-Yokoyama
Tokyo Medical and Dental University, Japan
Title: Glycosylation regulates CD38 assembly on the cell surface
Time : 14:10-14:30
Biography:
Miki Yokoyama received her Ph.D. in 1986 at the Dept. of Biophysics and Biochemistry, Faculty of Science, Tokyo University. She worked at the department of physiology in Nihon University School of Dentistry at Matsudo as a research associate (1986-1995) and as a lecturer (1995-2001). Then, she moved to the department of biochemistry in Tokyo Medical and Dental University (TMDU) and worked as a lecturer (2001-2004). Currently, she is an associate professor of the department. Her research interests include regulation of protein assembly on the membrane by glycosylation or lipid-environment.
Abstract:
Many proteins have their functions on the cell membranes or organelle membranes. To understand the function on the membranes, it is important to elucidate the cell-surface assembly. The leukocyte cell-surface antigen CD38 is a type II transmembrane glycoprotein and has four N-glycosylation sites. CD38 is the major NAD+ glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 also acts as a lipid raft-dependent signaling molecule to promote cell proliferation or death. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38, by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane ï¡-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers underlies the catalytic activity and the localization of CD38 in lipid rafts. The N-linked glycosylation sites are found to be located in strategic positions to prevent further self-association of the tetramer. Accordingly, the glycosylation is likely to ensure the function of CD38, by regulating the cell-surface assembly.
Chitra Mandal
CSIR-Indian Institute of Chemical Biology, India
Title: Sialylation of outer membrane porin protein D impedes β-lactam antibiotic entrance in Pseudomonas aeruginosa
Time : 14:30-14:50
Biography:
Prof. Chitra Mandal completed her Ph.D. at the age of 28 years from Indian Institute of Science, Bangalore and post doctoral studies at University of Pennsylvania. She is the Director of CSIR-Indian Institute of Chemical Biology, Kolkata, India and Sir J.C. Bose National Fellow, Head CSIR-Innovation Complex. She has published 150 research papers and holds 10 patents, guided 26 Ph.D students and transferred three technologies. She is an elected fellow of 'The World Academy of Sciences, main four Academies in India. Main theme of her group is to understand the mystery of glycosylation of biomolecules and their potential applications in health and diseases.
Abstract:
SIalic acids (Sias) are typically present as terminal sugars in oligosaccharide moieties attached to glycoproteins. Pseudomonas aeruginosa (PA), a gram-negative bacterium infects immuno-suppressed patients. We have established the presence of linkage-specific Sias on PA. Sialic acid-binding immunoglobulin-type lectins (Siglecs) are present on all immune cells. Sialylated PA (PA+Sias) binds to neutrophils through Sias-siglecs interactions. We have observed reduced oxidative burst, release of elastase and decrease NETs formation thus indicating subversion of host innate immunity. Next, we have affinity purified and sequenced twenty six sialoglycoproteins from PA+Sias. One such identified sialoglycoprotein is outer membrane porin protein D (OprD), a beta-barrel shaped channel-forming protein. To establish the role of Sias on OprD proteins, they are purified from sialylated (PA+Sias) and non-sialylated (PA-Sias) and their sialylation status are established. Profiling of glycan structures reveals the presence of sialylated N- and O-glycans in OprD+Sias. Bioinformatics studies reveal that amongst four N-glycosylation sites of OprD, Asn311 is present in the extracellular loop region having high solvent accessibility for its proper glycosylation. Core glycan moieties can properly fit into Asn311 site with no spatial overlaps with suitable glycosidic conformations. Molecular modeling studies suggest the presence of glycan structure with terminal bulky sialic acid hinders the channel passage of OprD towards β-lactam antibiotic permeabilization. This might be one of the new mechanisms for β-lactam antibiotic resistance of PA and thereby facilitating their survival in host. Our findings might help to open new avenue to design latestdrug which can enter cells freely even in presence of sialic acids.
Eliot T. Smith
East Tennessee State University, USA
Title: Expression of recombinant human mast cell chymase with asn-linked glycans in glycoengineered pichia pastoris
Time : 14:50-15:10
Biography:
Eliot T. Smith completed his PhD at the James H. Quillen College of Medicine at East Tennessee State University in 2013. He currently works as a postdoctoral researcher at the University of Pennsylvania, where he studies NDR kinases in the laboratory of Francis Luca.
Abstract:
Human mast cell chymase is a potent serine protease with roles in inflammation and allergy response. Chymase has received attention for its ability to generate angiotensin II from angiotensin I or angiotensin (1-12). The primary natural source of human chymase is skin tissue and recombinant expression provides a safe and abundant alternative for chymase research. One drawback of many expression systems, however, is the inability to generate proteins with human glycosylation patterns. To generate recombinant human chymase (rhChymase) with a glycosylation pattern that more closely resembles the natural enzyme, rhChymase was expressed and secreted in active form with (Man)5(GlcNAc)2 Asn-linked glycans using the SuperMan5 strain of GlycoSwitch® Pichia pastoris (BioGrammatics). Five milligrams of active enzyme were recovered from one liter of fermentation medium by cation exchange and heparin affinity chromatography. Purified rhChymase glycoprotein appeared as a single band migrating at an apparent molecular weight of 30 kDa on SDS-PAGE and treatment to remove glycosylation reduced the apparent molecular weight to 25 kDa, consistent with properties of the native enzyme. Western blotting with antibodies against human chymase labeled rhChymase. Active site titration with the potent chymase inhibitor Eglin C followed by kinetic analyses with peptide synthetic substrates demonstrated that glycosylated rhChymase possesses enzymatic activity that closely resembles its native counterpart. This work provides a source of active rhChymase with glycosylation similar to the as-yet unidentified human chymase glycan pattern(s) and offers a foundation for future production of chymase with true human glycosylation.