in silico studies of the bupag2
TRANSCRIPT
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Bhaskar Ganguly*, Ritu Shah, Shiv Prasadand H.P. Gupta
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PAG
1st isolated by Butler, 1982 in bovines from outerepithelial cell layer (chorion/ trophectoderm)
Expressed 7th day onwards
Ruminants have > 100 PAG genes
Expressed in pre-placental trophoblast, and post-implantation trophectoderm
Acts as local immunosuppresant
Also k/a Preg. Sp. Pr. B (PSPB), Preg. Sp. Pr. (PSP)-60
buPAG 1 is the major subfamily, PAG 2 is the majormember of the subfamily
Bubaline Pregnancy AssociatedGlycoprotein (buPAG)
PAGFamily
buPAG
1
buPAG
2
buPAG
8
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STEP 5
SEQUENCE ANALYSIS
cDNA Amino Acid
SEQUENCE
STRUCTURAL & FUNCTIONALANNOTATION
PHYSICO-CHEMICALCHARACTERISATION
STRUCTURE DETERMINATION
STEP 1
STEP 2
STEP 3
STEP 4
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STEP 1
TRANSLATE
Cloning using PAG2 specific primers
& Sequencing
GenBank Accession:
GU433184.1
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STEP 2
Number of amino acids: 99
Amino acid composition:Ala 4.0% Arg 2.0% Asn 4.0% Asp 6.1% Cys 3.0% Gln 3.0%Glu 2.0% Gly 11.1% His 1.0% Ile 10.1% Leu 7.1% Lys 2.0%Met 1.0% Phe 9.1% Pro 7.1% Ser 12.1% Thr 7.1% Trp 1.0%
Tyr 3.0% Val 4.0%
Number of negatively charged residues (Asp + Glu): 8Number of positively charged residues (Arg + Lys): 4
Atomic composition: C483H725N117O145S4Total number of atoms: 1474
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STEP 3
3D-JigSaw
ESyPred
SwissModel
TASSER
Structural Analysis &
Verification Server
(SAVES)
Quality Factorranged about 38 %(ESyPred) to 82 %
(SwissModel)
Quality Factorgreater than 85 %
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buPAG.pdb(TASSER)
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STEP 4
Molecular weight: 10619.0 (10.6 kDa) (Poor antigen)
Theoretical pI: 4.40
Extinction coefficients: (280 nm; measured in water)997010095 M-1cm-1 (Cys reduced Cystine)Absorbance (0.1% =1 g/l): (280 nm; measured in water)
0.939 - 0.951 (Cys reduced Cystine)
Estimated half-life:4.4 hrs (mammalian reticulocytes, in vitro) (Poor antigen)
Instability index: 64.70 (Unstable) (Poor antigen)Aliphatic index: 82.73
Grand avg. hydropathicity (GRAVY): 0.230 (Poor antigen)
ProtParam
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Secondary structure
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Front view Left view
Top view
STEP 5
TASSER
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hypothetical Ligand
probable Active Residues
Six active residues;four on surface, twowithin
One prominentligand binding siteon surface of themolecule
TASSER
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probable Active Residues
Four surface active
residues:
Ala 27
Ser 29
Lys 89
Ile 92
One prominent
ligand binding site
on surface of the
molecule
ligand bound to molecular surface TASSER
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PdbSum
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PdbSum
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3DLigandSite
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The bubaline Pregnancy Associated
Glycoprotein (buPAG) is:
Inducible,
Extracellular,
Aspartic Endopeptidase, and
Involved in intermediary metabolism.
ConFunc
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Mechanism of action of Aspartic Endopeptidases
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Mapping the catalytic site
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Mapping the catalytic site
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Mapping the catalytic site
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Mapping the catalytic site
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Mapping the catalytic site
Asp 90Asp 96
C atom ofpolyD
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SIGNIFICANT FINDINGS
Physical and bio-chemical properties were predicted
Characteristics for immunological optimization were
identified
3-D model of the protein was obtained
Structural characteristics of the protein were identified
The active catalytic site was mapped to its molecular
detail
The study shall make possible the optimization of
inhibitors for homologous molecules to allow a
physiological dissection of the exact role of buPAG 2
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