Target-GDP-mannose+pyrophosphorylase(GDP-MP)+(Leishmania+infantum)


 * Target (protein/gene name):** GDP-mannose pyrophosphorylase(GDP-MP)
 * NCBI Gene # or RefSeq#:** [|TritrypDB:LinJ.23.0120] **;** [|CAM68115.1]
 * Protein ID (NP or XP #) or Wolbachia#:** [|#5069112: XP_001465689.1]

Leishmaniasis infantum is known to cause Visceral leishmaniasis, a serious form of leishmaniasis that is a vector borne disease by intramacrophage protozoa via a sandfly called //Phlebotomus//. It can be caused by more than 20 leishmanial species. Leishmaniasis infantum is mainly found in the Latin America and the Mediterranean areas. Symptoms include high fever, weight loss, anaemia, and swelling of the spleen, and if left untreated the fatality can be as high as 100% [1]. It occurs worldwide, and an estimated 12 million people are infected as well as 350 million are at risk for the infection. Current treatment that people rely on are the pentavalent antimonial drugs that have been in use since the 1930s. Since this drug has been used for so long, drug resistance has become a recurring problem. Therefore, a new drug and specific drug to fight this disease is needed [2].
 * Organism (including strain):** //Leishmania infantum//
 * Etiologic Risk Group (see link below):** Apprendix B-II-C. Risk Group 2(RG2) - Parasitic Agents
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**
 * Essentiality of this protein:** Essential

GDP-mannose pyrophosphorylase (GDP-MP) is an enzyme that plays a role in glycosylation and vital for amastigote survival [3]. //Leishmania// protozoa synthesize a variety of mannose-containing glycoconjugates that are crucial for virulence in the sandlfly vector and mammalian host. For synthesis to occur, GDP-MP must catalyze a mannose-1-phosphate to create an activated mannose donor, GDP-mannose. GDP-MP has been shown to self-associate to an enzymatically active and stable hexamer that dissociates to trimmers and monomers at low protein concentrations, low ionic strength, and at alkaline pH. Immunofluorescence and subcellular fractionation showed that GDP-MP is a cytoplasmic protein. Studies has also shown that Leishmania parasites that lack the enzyme are completely avirulent [2]. An in silico analysis has also shown the human and parasite GDP-MP 3D homology models were similar. However, there was a motif specific to the catalytic site of the parasite enzyme, signifying that it could be used to specifically inhibit the parasite enzyme without affecting the human homolog [3]. This as well as GDP-MP’s other attributes indicates that GDP-MP is a potential drug candidate for //Leishmaniasis//.


 * Complex of proteins?:** Yes:  α-D-mannose 1-phosphate + GTP + H +  ↔ GDP-α-D-mannose + diphosphate
 * Druggable Target:** Yes

[]
 * *EC#: ** 2.7.7.13
 * Link to BRENDA EC# page:**
 * --** Show screenshot of BRENDA enzyme mechanism schematic

[] [|Davis, A.J.; Perugini, M.A,; Smith, B.J.; Stewart, J.D.; Ilg, T.; Hodder,A.N.; Handman,E., Properties of GDP-mannose Pyrophosphorylase, a Critical Enzyme and Drug Target in Leishmania Mexicana. Journal of Biological Chemistry 2004, 279, 12462-12468.] -- PDB # or closest PDB entry if using homology model: PDB strucutre 2X65: CRYSTAL STRUCTURE OF T. MARITIMA GDP-MANNOSE PYROPHOSPHORYLASE IN COMPLEX WITH MANNOSE-1-PHOSPHATE. ([|2X65]) -- For Homology Model option:
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link (or citation) to paper that contain****s assay i****nformation:**
 * Structure Available (PDB or Homology model)**

Sequence ID: lcl|58401Length: 336Number of Matches: 2

Related Information

Range 1: 2 to 109 Graphics Next Match Previous Match


 * Alignment statistics for match #1 ||
 * **Score ** ||  **Expect **  ||  **Method **  ||  **Identities **  ||  **Positives **  ||  **Gaps **  ||
 * 30.8 bits(68) || 4e-05 || Compositional matrix adjust. || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">32/115(28%) || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">52/115(45%) || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">12/115(10%) ||

Query 9 MRAVILVGGFGTRLRPL-TLTTPKPLVP-FCNKPMIIHQIEALKAVGVTEVILAVAYRP- 65

M+A+IL GG G R PL T TPK + F NK ++ E + + ++ V ++

Sbjct 2 MKALILAGGSGERFWPLSTPETPKQFLKLFGNKSLMRWTFERVLEEMDPKDVIVVTHKDY 61

Query 66 -EAMKEQMDEWSRKLGVSFVFSVEEEPL-GTAGPLALARDILMQDDKPFFVLNSD 118

E K+++ E + ++ EP+ P L DD+P VL +D

Sbjct 62 VERTKKELPELPDE---NIIAEPMKKNTAPACFIGTKLADDDEPVLVLPAD 109

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Range 2: 59 to 76 Graphics Next Match Previous Match [|First Match]


 * <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: center;">Alignment statistics for match #2 ||
 * **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Score ** ||  **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Expect **  ||  **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Method **  ||  **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Identities **  ||  **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Positives **  ||  **<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Gaps **  ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">16.9 bits(32) || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">0.89 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Compositional matrix adjust. || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">7/18(39%) || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">9/18(50%) || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">0/18(0%) ||

Query 231 KDYILGMTKFIPSLVHGN 248

KDY+ K +P L N

Sbjct 59 KDYVERTKKELPELPDEN 76

Query Coverage: 33% Max % Identities: 39% % Positives: 45% & 50% Chain used for homology: Both chains were used for alignment.
 * PDB strucutre 2X65 had 2 chains; the pairwise alignment for both chains are shown above.

GDP-MP was subcloned into the expression vector pMALc2T, with an N-terminal MBP tag and expressed in //E.coli//. BL21(DE3)pLysS cells that contain GDP-MP were made to inoculate LB broth containing chloramaphenicol, ampicillin, and glucose. Expression was stimulated by adding isopropyl-β-D-thiogalactopyranoside and harvested by centrifigution.The culture was then resuspended in a column buffer consisting of Tris-HCL, NaCl. EDTA, and dithiothreitol. After the cells were thawed and sonicated, a soluble lysate was collected after centrifugation for purification.
 * Current Inhibitors:**
 * Expression Information (has it been expressed in bacterial cells):** Expression has been done in //E.coli//, but from //L. mexicana.//
 * Purification Method:**

Purification of the lysate was done by affinity chromotgraphy on amylose resin. The fractions containing the protein were pooled against thrombin hydrolysis buffer that was consisted of Tris, NaCl, CaCl2, DTT. Then the protein was digested with thrombin in order to remove the MBP fusion partner. Before further purification, the sample was separated overnight with Tris, NaCl, and DTT by using ion-exchange chromatography. Then, the protein was unbounded by loading the solution onto a HiTrap Q Sepharose HP column and eluted with NaCl. The eluted fractions were then analyzed by SDS-PAGE. [2].

MSASDGQGMRAVILVGGFGTRLRPLTLTTPKPLVPFCNKPMIIHQIEALKAVGVTEVILA VAYRPEAMKEQMDEWSRKLGVSFVFSVEEEPLGTAGPLALARDILMQDDKPFFVLNSDVT CTFPMQELLDFHKAHGGEGTIMVSQVTQWEKYGVVVYSPQNYQIERFVEKPSRFLGDRIN AGIYIFNKSILDRIPPRRTSIEKEIFPAMAAEGQLYAFNLEGFWMDVGQPKDYILGMTKF IPSLVHGNRETEQLHTEDMEHQRGGRFTVIGASLIDPSAKIGDGAVIGPYASIGANCVIG ESCRIDNAAILENSKVGKGTMVSRSIVGWNNRIGSWCHIKDISVLGDDVEVKDGVILIGT KVLPNKDVGEHRFEPGIIM ATGTCTGCATCCGATGGCCAGGGAATGCGGGCAGTGATTCTGGTCGGCGGCTTTGGCACT CGGCTCCGTCCGCTGACGCTGACGACGCCGAAGCCCCTCGTCCCATTTTGCAACAAGCCG ATGATCATTCACCAAATCGAGGCATTGAAGGCGGTCGGCGTAACGGAGGTGATTCTAGCT GTGGCGTACCGGCCGGAGGCGATGAAGGAGCAGATGGACGAGTGGTCACGGAAGCTTGGT GTTTCCTTCGTGTTCTCCGTCGAGGAGGAGCCGCTCGGCACGGCCGGCCCGCTTGCACTC GCCCGCGATATCCTGATGCAGGATGACAAGCCTTTTTTTGTGCTCAACTCCGATGTCACC TGTACGTTTCCGATGCAGGAGCTGCTGGACTTCCACAAGGCGCACGGCGGCGAGGGCACC ATCATGGTCTCGCAGGTGACGCAGTGGGAGAAGTACGGTGTCGTCGTCTACTCGCCGCAG AATTACCAGATCGAGCGGTTTGTGGAGAAGCCGAGCAGATTTCTTGGGGACCGAATCAAC GCCGGCATTTATATCTTCAACAAGAGCATCCTGGACCGCATTCCTCCCCGCCGAACATCC ATCGAGAAGGAGATCTTCCCTGCGATGGCCGCGGAGGGGCAGCTCTACGCATTCAACCTG GAGGGCTTCTGGATGGATGTCGGCCAGCCCAAGGACTACATCCTTGGTATGACCAAGTTC ATTCCGTCCTTGGTTCATGGGAACCGCGAAACGGAGCAGTTACACACGGAGGACATGGAA CACCAGCGTGGCGGCCGCTTCACTGTGATCGGCGCCTCTCTGATTGACCCCTCCGCAAAG ATCGGCGATGGCGCCGTGATTGGCCCCTATGCTTCGATTGGCGCCAACTGCGTTATTGGC GAGTCGTGCCGCATCGATAATGCTGCCATCTTGGAGAACTCCAAGGTCGGCAAGGGTACA ATGGTGTCTCGCAGCATTGTGGGCTGGAACAACCGTATCGGCAGCTGGTGCCACATCAAG GACATATCTGTGCTGGGCGATGACGTGGAGGTCAAGGATGGCGTGATTCTCATCGGCACC AAAGTGCTCCCCAACAAGGATGTCGGCGAGCACCGCTTCGAGCCTGGGATCATCATGTAG
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**
 * length of your protein in Amino Acids:** 379
 * Molecular Weight of your protein in kiloDaltons:** 41.84kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 39670 and 39420 M^-1cm^-1
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * CDS Gene Sequence (paste as text only):**


 * GC% Content for gene:** 58.33%
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**
 * GC% Content for gene (codon optimized):**

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * ( link to DNA Works output text file - **that should be saved in your Google Docs folder after you did the primer design protocol)


 * Primer design results for 'tail' primers (this is just 2 sequences):**
 * NCBI Gene # or RefSeq#:**
 * Protein ID (NP or XP #) or Wolbachia#:**
 * Organism (including strain):**
 * Etiologic Risk Group (see link below):**
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**
 * Essentiality of this protein:**
 * Complex of proteins?:**
 * Druggable Target:**


 * References:**
 * 1) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R.W.; Alvar, J.; Boelaert, M., Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?. //Nature Reviews// **2007**, 5, 873-882.
 * 2) Davis, A.J.; Perugini, M.A,; Smith, B.J.; Stewart, J.D.; Ilg, T.; Hodder,A.N.; Handman,E., Properties of GDP-mannose Pyrophosphorylase, a Critical Enzyme and Drug Target in //Leishmania Mexicana//. // Journal of Biological Chemistry // **2004**, 279, 12462-12468.
 * 3) Pomel, S.; Rodrigo, J.; Hendra, F.; Cave, C.; Loiseau, P.M., In silico analysis of a therapeutic target in Leishmania infantum: the guanosine-diphospho-D-mannose pyrophosphorylase. //Parasite// **2012**, 19, (1), 63-70.