Research Article: Conformational Differences between Active Angiotensins and Their Inactive Precursors

Date Published: , 2012

Publisher: A.I. Gordeyev

Author(s): O.N. Solopova, L.P. Pozdnyakova, N.E. Varlamov, M.N. Bokov, E.V. Morozkina, Т.А. Yagudin, P.G. Sveshnikov.

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Abstract

The peptide conformation in the context of a protein polypeptide chain is
influenced by proximal amino acid residues. However, the mechanisms of this
interference remain poorly understood. We studied the conformation of
angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion
from a precursor protein angiotensinogen and contain an identical peptide core
structure. Using the example of angiotensins 1, 2 and 3, it was shown that
similar amino acid sequences may have significant conformational differences in
various molecules. In order to assess the conformational changes, we developed a
panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and
3 and studied their cross-reactivity in indirect and competitive ELISAs. It was
found that the conformations of inactive angiotensin1 and the corresponding
fragment of angiotensinogen are similar; the same is true for the conformations
of active angiotensins 2 and 3, whereas the conformations of homologous
fragments in the active and inactive angiotensins differ significantly.

Partial Text

Since the appearance of the hybridoma technology in 1975 [1], a large number of monoclonal antibodies to various
substances have been obtained. Despite this, new antibodies are still highly sought;
namely, antibodies with particular properties, antibodies to particular epitopes and
to newly discovered proteins and other organic and inorganic compounds. Normally, it
is possible to obtain new proteins only in limited amounts, and frequently it is
very difficult or even impossible to isolate them in their pure form with the
natural conformation preserved. These kinds of proteins cannot be used for
immunization to obtain antibodies; hence, in the majority of cases, the only viable
option is immunization with synthetic peptides corresponding to particular fragments
of the desired protein. Along with the apparent advantages, this approach also has a
number of shortcomings: i.e. , peptides, when found in proteins,
have a significantly lower degree of freedom than when they are in a free state. As
a result, the antibodies against peptides are not always capable of binding to
full-size proteins [2].

In this work human recombinant angiotensinogen (“Sigma”, USA),
angiotensins 1, 2 and 3 (“American Peptide”, USA), recombinant Hsp70
from Mycobacterium tuberculosis obtained in our laboratory [5], BALB/c mice, and the mouse myeloma cell line
Sp2/0 were used.

The human and mouse angiotensins 1, as well as angiotensins 2 and 3, have identical
amino acid sequences [13]. In addition,
angiotensins 2 and 3 exhibit physiological activity and their introduction into the
body at doses needed for immunization (10–50 µg/mouse) leads to a fatal
outcome even in the case of intramuscular and subcutaneous injections. Altogether,
it makes angiotensins extremely inconvenient immunogens; however, their conjugation
with Hsp70, an adjuvant protein from M. tuberculosis, allowed to
overcome the immunological tolerance and to eliminate the toxicity. As a result, we
obtained monoclonal antibodies against each angiotensin.

Summarizing the results, we can draw the following conclusions:

 

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