X. Long Zheng

Publications (62) View all

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  Article: Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura.

  X Zheng, D Chung, T K Takayama, E M Majerus, J E Sadler, K Fujikawa
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  ABSTRACT: Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn(2+) and Ca(2+) ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the protein at different positions after the protease domain. Alternative splicing may have functional significance, producing proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.
  Journal of Biological Chemistry 12/2001; 276(44):41059-63. · 4.77 Impact Factor
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  Article: Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

  D Lu, K Fütterer, S Korolev, X Zheng, K Tan, G Waksman, J E Sadler
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  ABSTRACT: Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.
  Journal of Molecular Biology 10/1999; 292(2):361-73. · 4.00 Impact Factor
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  Article: Differential activity of diethylstilbestrol versus estradiol as neonatal endocrine disruptors in the female hamster (Mesocricetus auratus) reproductive tract.

  W J Hendry, B L DeBrot, X Zheng, W S Branham, D M Sheehan
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  ABSTRACT: The synthetic estrogen diethylstilbestrol (DES) is a potent neonatal endocrine disruptor in the hamster. To test the specificity of this phenomenon, newborn animals were treated with 100 microgram of either DES or the natural estrogen, estradiol-17beta (E2). Of the two, neonatal DES exposure caused greater morphological disruption throughout the female reproductive tract in prepubertal animals and in adults that either retained their ovaries or were ovariectomized and then given the same levels of chronic E2 stimulation. In the uterus, a characteristic histopathological profile, including enhancement of both hyperplastic and apoptotic activity, was initiated prepubertally and exclusively in the endometrial epithelial cell compartment from the neonatally DES-treated animals and then was promoted by E2 stimulation during adulthood. Interestingly, apoptotic activity was not detected in an area of endometrial epithelium that progressed to the neoplastic state in a DES-exposed animal. Lastly, chronic estrogen induction of lactoferrin was also restricted to the DES-exposed endometrium. We conclude that 1) DES is more active than E2 as a perinatal endocrine disruptor in the hamster and 2) this experimental system should be generally useful as a means to screen compounds for such activity and then probe their mechanism of action.
  Biology of Reproduction 08/1999; 61(1):91-100. · 4.01 Impact Factor
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  Article: Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts.

  X Zheng, D Lu, J E Sadler
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  ABSTRACT: Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.
  Journal of Biological Chemistry 02/1999; 274(3):1596-605. · 4.77 Impact Factor
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  Article: Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice.

  J Xue, Q Wu, L A Westfield, E A Tuley, D Lu, Q Zhang, K Shim, X Zheng, J E Sadler
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  ABSTRACT: Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2(-/-) embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2(-/-) embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2(-/-) neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.
  Proceedings of the National Academy of Sciences 07/1998; 95(13):7603-7. · 9.68 Impact Factor