Xiaotian Zhong

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor‐bound enzymes exhibit two essentially different catalytic‐site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

The Structure of the Lingo-1 Ectodomain, a Module Implicated in Central Nervous System Repair Inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200Å2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.

N‐linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression

P2Y12 receptor is a Gi‐coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N‐linked glycosylation sites at its extra cellular amino‐terminus, which may modulate its activity. Studies of both tunicamycin treatment and site‐directed mutagenesis have revealed a dispensable role of the N‐linked glycosylation in the receptors surface expression and ligand binding activity. However, the non‐glycosylated P2Y12 receptor is defective in the P2Y12‐mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N‐linked glycans in receptors signal transducing step but not in surface expression or ligand binding.

A YEAST GOLGI E-TYPE ATPASE WITH AN UNUSUAL MEMBRANE TOPOLOGY

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca2+, Mg2+, and Mn2+. The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH2-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH2-terminal portion of the protein and no NH2-terminal cleavage of the protein is observed, demonstrated by the detection of NH2-terminal tagged Apy1p, the NH2-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH2 terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.

Regulation of Yeast Ectoapyrase Ynd1p Activity by Activator Subunit Vma13p of Vacuolar H+-ATPase

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H+-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Δmutant was drastically increased compared with that of Ynd1p inVMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Δ mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.

Regulation of secreted Frizzled-related protein-1 by heparin.

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.

H6PDH interacts directly with 11β-HSD1: Implications for determining the directionality of glucocorticoid catalysis

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11beta-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11beta-HSD1, and with cell lysates expressing H6PDH and 11beta-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11beta-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11beta-HSD1 physically as well as modulate 11beta-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11beta-HSD1 activity.

ATP Uptake in the Golgi and Extracellular Release Require Mcd4 Protein and the Vacuolar H+-ATPase

Extracellular nucleotides signal via a large group of purinergic receptors. Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown. We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae. This release occurred through the vesicular trafficking pathway initiated by ATP uptake into the Golgi compartment. Both the compartmental uptake and the extracellular release of ATP were regulated by the activity of the vacuolar H+-ATPase. It is likely that the Mcd4p pathway is generally involved in non-mitochondrial ATP movement across membranes, it is essential for Golgi and endoplasmic reticulum function, and its occurrence led to the appearance of P2 purinergic receptors.

Swift residue-screening identifies key N-glycosylated asparagines sufficient for surface expression of neuroglycoprotein Lingo-1.

Advances in genomics and proteomics have generated the needs for the efficient identification of key residues for structure and function of target proteins. Here we report the utilization of a new residue‐screening approach, which combines a mammalian high‐throughput transient expression system with a PCR‐based expression cassette, for the study of the post‐translational modification. Applying this approach results in a quick identification of essential N‐glycosylation sites of a heavily glycosylated neuroglycoprotein Lingo‐1, which are sufficient for the support of its surface expression. These key N‐glycosylated sites uniquely locate on the concave surface of the elongated arc‐shape structure of the leucine‐rich repeat domain. The swift residue‐screening approach may provide a new strategy for structural and functional analysis.

Recent Advances in Glycosylation Modifications in the Context of Therapeutic Glycoproteins

Glycosylation is one of the most complex post-translation modifications, commonly found in many cell surface and secreted eukaryotic proteins. 1-2% of the human transcriptome encodes proteins that link to glycosylation. Many protein-based biotherapeutics approved or in clinical trials are glycoproteins. The oligosaccharides covalently attached to therapeutic glycoproteins pose biological benefits as well as manufacturing challenges. The present chapter reviews the structure and function of glycosylation, glycoform patterns observed for the biotherapeutic proteins produced by various host systems, and analytic methods for the characterization of glycoforms. Recent advances in utilizing glycosylation as a strategy to improve biotherapeutics properties are also discussed.