Bhaskar Ganguly*, Ritu Shah, Shiv Prasad

and H.P. Gupta

 Bubaline Pregnancy Associated
Glycoprotein (buPAG)
• PAG – 1st isolated by Butler, 1982 in bovines from outer
epithelial 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 major
member of the subfamily buPAG
2
buPAG buPAG
1 8

PAG
Family
 cDNA → Amino Acid
STEP 1
SEQUENCE

STEP 2 SEQUENCE ANALYSIS

STEP 3 STRUCTURE DETERMINATION

PHYSICO-CHEMICAL
STEP 4
CHARACTERISATION

STRUCTURAL & FUNCTIONAL

STEP 5
ANNOTATION
STEP 1
Cloning using PAG 2 specific primers
& Sequencing

GenBank Accession:
GU433184.1

TRANSLATE
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): 8
Number of positively charged residues (Arg + Lys): 4

Atomic composition: C483H725N117O145S4

Total number of atoms: 1474
STEP 3

3D-JigSaw

Quality Factor
ranged about 38 %
ESyPred (ESyPred) to 82 %
(SwissModel)
Structural Analysis &
Verification Server
(SAVES)
SwissModel

Quality Factor
TASSER greater than 85 %
buPAG.pdb
(TASSER)
STEP 4

Molecular weight: 10619.0 (10.6 kDa) (Poor antigen)

Theoretical pI: 4.40
Extinction coefficients: (280 nm; measured in water)
9970 – 10095 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
Hydrophobicity Plot (Kyte Doolittle)
Secondary structure
STEP 5

Front view Left view

Top view TASSER

Six active residues;
four on surface, two
within

One prominent
ligand binding site
on surface of the
molecule

≡ probable Active Residues

≡ hypothetical Ligand
TASSER
 Four surface active
residues:
• Ala 27
• Ser 29
• Lys 89
• Ile 92

≡ probable Active Residues

One prominent
ligand binding site
on surface of the
molecule

TASSER ligand bound to molecular surface ≡

PdbSum
PdbSum
3DLigandSite
The bubaline Pregnancy Associated
Glycoprotein (buPAG) is:

• Inducible,

• Extracellular,

• Aspartic Endopeptidase, and

• Involved in intermediary metabolism.

ConFunc
Mechanism of action of Aspartic Endopeptidases
Mapping the catalytic site
Mapping the catalytic site
Mapping the catalytic site
Mapping the catalytic site
C atom of
polyD

Asp 90

Asp 96

Mapping the catalytic site

 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