In silico studies of the buPAG2

8/7/2019 In silico studies of the buPAG2

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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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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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Date post:	09-Apr-2018
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In silico studies of the buPAG2

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