Variable region gene expression and structural motifs of human polyreactive immunoglobulins
- Publication Type:
- Thesis
- Issue Date:
- 1997
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Polyreactive immunoglobulins (Ig) exhibit a capacity to recognise multiple,
structurally dissimilar antigens through a single combining site. This characteristic
differentiates these Igs from monoreactive Igs which bind to a single antigen, usually
with high specificity and affinity. Chronic B lymphocytic leukaemia (B CLL) is a
malignancy identified by the incessant accumulation, in the peripheral circulation, of B
lymphocytes of a mature and resting morphology. B CLL malignant cells generally
express both surface IgM and the pan T cell antigen CD5. Moreover, the IgM on the
surface of these CD5 positive B CLL cells is frequently polyreactive. This thesis
examines the structural diversity found in the combining sites of B CLL derived Igs in
an attempt to elucidate the structural basis of polyreactive antigen binding displayed by
a significant proportion of human Igs. The genes encoding the variable (V) domains
of five B CLL derived IgM antibodies (Bel, Tre, Yar, Hod and Jak) were cloned and
sequenced (Chapter Two). When the light chain V domain genes were aligned with
the closest germline VL and JL coding DNA sequences it was determined that there
was either a complete absence of somatic mutation (Tre, Yar and Jak) or a minimal
number of mutations (Bel and Hod) present in the rearranged VL domain genes. A
remarkable fidelity in the splicing of VL to JL genes was noted suggesting that the
diversity, normally introduced through variability of splicing VL to JL, is reduced in
Igs expressed by B CLL cells. Furthermore, the markedly reduced primary structural
diversity was highlighted when two of the VL domain genes (Yar and Hod) were
found to be different in sequence by only four nucleotides and two amino acids. The
heavy chain V domain genes of the same five Igs were sequenced in another study
(Brock, 1995), however, it was interesting to analyse the sequences of the VH domain
genes and compare them with the VL domain genes. The naive or gerrnline nature of
the B CLL antibodies was reflected in the VH genes by either an absence or a low
frequency of mutations within these sequences compared with germline
immunoglobulin gene sequences. No obvious conserved motif, which could be
related to polyreactivity, was observed when the primary protein sequence was
analysed for distribution of identical or similar amino acids. Thus, homology
modelling was used to construct three-dimensional models of the Fv (VL-VH)
portions of the five B CLL IgM molecules to examine the structures of the combining
sites of these Igs (Chapter Three). Framework regions were constructed using X-ray
coordinates taken from highly hon~ologous human variable domain structures.
Complementarity determining regions (CDR) were predicted by grafting loops, taken
from known Ig structures, onto the Fv framework models. The CDR templates were
selected, where possible, to be of the same length and of high residue identity or
similarity. If a single template CDR was not appropriate to model a particular CDR the
loop was built from loop sterns of known conformation, followed by chain closure
with a p-turn. Template models were refined using standard molecular mechanics
simulations. The binding sites were either relatively flat or contained a deep cavity at
the VL-VH domain interface. Further differences in topology were the result of some
CDR loops protruding into the solvent. Examination of the electrostatic molecular
surface did not reveal a common structural feature within the binding sites of the five
polyreactive Fv. While two of the binding cavities were positively charged the other
three structures displayed either negatively charged or predominantly hydrophobic
combining sites. These findings suggested that a diversity of structural mechanisms
are involved in polyreactive antigen binding. Rcsidues within CDRs which have
aromatic side-chains and are partially exposed to solvent were distributed across large
regions of the combining sites. It is possible that these aromatic residues are
responsible for the conserved binding to mouse Igs observed (Chapter Two) for the B
CLL derived polyreactive IgM molecules. Two Fv molecules (Be1 and Tre) were
cloned as dicistronic constructs, into the bacterial expression vector pFLAG. The
expression of the Fvs was fully characterised and unfortunately the VL and VH of Be1
and Tre Igs did not associate in an appropriate manner to yield large quantities of
purified Fv (Chapter Four). Expression of correctly folded and stabilised fragments
of human polyreactive immunoglobulins would enable the structural basis for the
polyreactive binding phenomenon to be fully explored using protein crystallography.
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