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Structure, interaction and design studies involving regulatory peptides and proteins |
| Principal Investigator : Dinakar M Salunke
Project Associates
/ Assistants Technical Officer Ph D Students Collaborators The
structural aspects of molecular recognition and its applications in analyzing
the mechanisms associated with specific regulatory events and in rational
molecular design is the theme of research. The objectives of the project are (i)
understanding the protein architecture, (ii) analysis of the structural
principles of molecular recognition and mimicry, (iii) structural biology of
various regulatory events and (iv) rational molecular design studies based on
the above. As
reported in the previous report, anti-mannopyranoside monoclonal antibodies
that could also recognize the carbohydrate mimicking peptides have been
generated. A total of about 50 clones were found to be positive for
recognizing the mannopyranoside antigen. Both, the clones showing
cross-reactivity to the 12mer peptide or recognizing only the carbohydrate
antigen were present, though the latter were only about 10% of the total anti-mannopyranoside
clone population. A panel of four IgG antibodies named 1H7, 2D10, 1H11 and 5F5
were selected for further studies. Of these antibodies, 2D10, 1H11 and 5F5
show crossreactivity to the peptide while the 1H7 antibody shows binding to
the mannopyranoside ligand only. Among the antibodies, 1H7, 1H11 and 5F5, are
of IgG1 isotype while the 2D10 displays IgG2b isotype. The
binding kinetics of these antibodies for both the ligands were studied by
surface plasmon resonance based measurements at ambient temperature.
Conjugates of the mannopyranoside and 12mer peptide with BSA were used as
immobilized ligands and each one of the antibodies were studied for their
binding to the mannopyranoside and the peptide ligands. The association and
the dissociation rate constants were determined and used for deriving the
dissociation constants of all antibodies for their respective ligands. Among
the various carbohydrate-binding antibodies, 1H7 has the highest affinity (KD=0.2mM)
for the mannopyranoside. 2D10 shows slightly higher affinity of KD=0.9uM
for the carbohydrate as compared to that for the peptide ligand (KD=8.8mM).
Both 1H11 and 5F5 show highly comparable affinity for mannopyranoside although
1H11 shows higher affinity for the peptide than for the mannopyranoside. The
four different antibodies not only show different ligand affinities for the
peptide and carbohydrate ligands but also show differences in the kinetics of
binding to the two ligands. We therefore wanted to further explore the
diversity among these antibodies at the gene level. To determine the germline
origin and relatedness among the four antibodies, the nucleotide sequencing
for both the light and the heavy chains of these antibody clones were carried
out. The nucleotide sequences thus obtained were used to search for the
germline origins of the antibodies from the database using Ig-BLAST. The
nucleotide sequence of the light chain of the 1H7 antibody shows homology
(97%) to ce9, the V region germline gene sequence and JK1, the J region
germline sequence. 2D10 antibody is highly homologous (97%) to the 21-12
variable germline gene sequence and JK2. The light chain of 1H11 antibody
shows identity to the 19-15 (99%), V region germline gene sequence and JK4.
The 5F5 antibody shares homology (99%) with the bb1, V region germline gene
sequence and JK2 sequence. The heavy chains of all the four antibodies
originate from the various gene members of the J558 family. Heavy chain of 1H7
antibody is derived from the J558.17 showing sequence homology (97%) to this V
region germline gene sequence and to D element, DSP2.2, and JH2 germline
sequence. 2D10 heavy chain has high sequence identity (93%) to the VH
gene J558.12 and also shows homology to D element gene DFL16.1 and JH2. 1H11
shows sequence homology (95%) to the J558.44 germline sequence and to DSP2.9
and JH3 gene elements. The heavy chain of 5F5 antibody shows homology to
J558.8 (98%) germline member and to DQ52, the D-element sequence and JH4
sequence. The combination of different DH
and JH
elements to the various members of the J558 VH
chain as seen in the case of four anti-mannopyranoside antibodies suggests
that the four clones have independent B-cell origin. The variability seen in
the light chain gene usage reinforces this view, that the four clones are not
the product of somatic mutations from a given B-cell. Thus, a variety of gene
arrangements can probably give rise to the mannopyranoside recognizing
paratopes but these may differ in their recognition specificity for both, the
carbohydrate as well as the 12mer peptide, epitopes. The
YPY motif of the crossreacting peptide has been implicated in
mimicking a variety of other carbohydrates, we therefore wanted to analyze if
the various antibodies recognizing the 12mer peptide would cross-react with
any other carbohydrate ligands. Since these antibodies show variable
affinities for the mannopyranoside ligands, we also wanted to see if they
differed in their specificity profiles as well. For this, competition based
assays were carried out where various sugars in solution were made to compete
for binding to the antibody as against the immobilized mannopyranoside or the
peptide ligand. It is evident that the three mimicry recognizing peptides do
not show much differentiation between the three sugars, whereas 1H7, the only
carbohydrate recognizing antibody, shows much higher affinity for the
mannopyranoside as compared to glucopyranoside and lactose. It is interesting
to note that polyclonal antibodies have earlier been shown to recognize
glucopyranoside with lower affinity than mannopyranoside, while lactose shows
minimal competition and behaves as a non-specific sugar. Similar degenerate
specificity is shown for various sugars by the three peptide recognizing
antibodies when sugars were competing against the immobilized 12mer peptide.
In contrast to the polyclonal antibody cross-reactivity data where the sugars
were competing against the immobilized mannopyranoside ligands, the polyclonal
data in case of immobilized peptide behaves exactly similar to the three
antibodies. As
part of another on-going activity concerning structural basis of carbohydrate
mimicry, we had earlier determined the crystal structure of concanavalin A
bound to a porphyrin analog, meso-tetrasulphonatophenylporphyrin (H2TPPS),
and established the mimicry of mannopyranoside and porphyrin in terms of the
interactions with the lectin. We have now determined and refined the crystal
structure of H2TPPS
with jacalin at 1.8Å resolution and in the process of refining the structure
in complex with peanut lectin at 2.9Å resolution. In
the crystal structure of H2TPPS-jacalin
complex, a pair of stacked porphyrins interact with two jacalin molecules,
both of which belong to two independent jacalin tetramers. This dimer of
porphyrins with a monomer of jacalin forms the asymmetric unit of the present
crystal structure. Thus, a dimer of porphyrin molecules is sandwiched between
two symmetry related jacalin monomers. This leads to extensive non-covalent
cross-linking of the jacalin molecules in the crystal structure. The two
porphyrins do not interact at the similar site with the jacalin molecules. One
of them exhibits specific interactions at the carbohydrate binding site on
jacalin, while the other interaction is largely resulting while facilitating
crystal contacts. The binding of the porphyrin molecule does not seem to cause
any major changes in the backbone conformation of the jacalin molecule. The two porphyrin molecules are staggered with respect to each other such that the porphine cores of the two are not eclipsed but seem to be translated with respect to each other, unlike in case of H2TPPS-ConA complex. Seven water molecules are also found within the van der Waals distance of this porphyrin molecule. The nitrogen atom N8 of the porphine ring forms a hydrogen bond with a water molecule. Thus the nature of interactions involving H2TPPS with jacalin are substantially different from those with ConA although both cases show water mediated bonding with the protein. The preliminary studies in case of peanut lectin and H2TPPS suggests yet another mode of binding of H2TPPS with this lectin. Publications Original
peer-reviewed articles 1.
Nair DT, Kaur KJ, Singh K, Mukherjee P, Rajagopal D, George A, Bal V,
Rath S, Rao KV and Salunke DM (2003) Mimicry of native peptide antigens by the
corresponding retro-inverso analogs is dependent on their intrinsic structure
and interaction propensities. J Immunol 70:1362-1373. 2.
Nagpal S, Kaur KJ, Jain D and Salunke DM (2002) Plasticity in structure and
interactions is critical for the action of indolicidin, an antibacterial
peptide of innate immune origin. Protein Sci 11:2158-2167. 3.
Chakraborty S, Chakraborty N, Jain D, Salunke DM and Datta A (2002) Active
site geometry of oxalate decarboxylase from Flammulina velutipes: Role of
histidine coordinated manganese in substrate recognition. Protein Sci 11:2138-2147. 4.
Manivel V, Bayiroglu F, Siddiqui Z, Salunke DM and Rao KVS (2002) The primary
antibody repertoire represents a linked network of degenerate
antigen-specificities. J Immunol 169:888‑897. 5.
Gangatirkar P, Gangadharan S, Narendranath A, Nagpal S, Salunke DM and Karande
AA (2002) Monoclonal antibodies to gonadotropin-releasing hormone (GnRH)
inhibit binding of the hormone to its receptor. Hybrid Hybridomics 21:281-286. 6.
*Hemachand T, Gopalakrishnan B, Salunke DM, Totey SM and Shaha C (2002) Sperm
plasma membrane associated glutathione S- transferases as gamete recognition
molecules. J Cell Sci 115:2053-2065 (*in press last year, since
published). |