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Identification of a Human Milk Glycoprotein that Binds DC-SIGN and Inhibits HIV-1 Transfer to CD+ T Lymphocytes
Marloes Naarding*1, A Dirac2, D Speijer1, G Pollakis1, and W Paxton1
1Academic Med Ctr, Univ of Amsterdam, The Netherlands and 2Netherlands Cancer Inst, Amsterdam
Background:
DC-SIGN-expressing cells can capture HIV-1 and transfer virus to CD4+
lymphocytes. We have demonstrated that human milk can inhibit the interaction between DC-SIGN
and gp120 and that the inhibitory effect can be alleviated by pre-incubation of
human milk with an antibody directed against the saccharide Lewis X (LeX)
motif. Polyvalent linked-LeX and BSA-LeX mimicked the
inhibition of human milk while LeX trisaccharide did not, suggesting
that a glycoprotein(s) in human milk is responsible.
Methods: The
Raji-DC-SIGN-expressing cell line and a DC-SIGN-Fc binding ELISA were utilized
to monitor inhibition of viral transfer to CD4+ lymphocytes and
DC-SIGN binding, respectively. Western blot analysis was performed using a LeX
specific Ab and a DC-SIGN-Fc construct. MALDI technology was used to analyze
the protein isolated from SDS-page gels. Human milk was size fractionated,
boiled, and trypsin digested.
Results: When
tested in the binding and transfer experiments the human milk compound was
found to be heat resistant, trypsin resistant, and of a specific molecular
weight, indicating that not all LeX-carrying proteins provide for
viral inhibition. From studying human milk samples of different mothers, we
showed variable inhibitory activities in both assays. Both LeX Ab
and DC-SIGN-Fc stained Western blots showed down-regulation of 1 specific band
for weak-binding mothers compared with strong binders. The band of the weak
binders and the corresponding band of strong binding mothers was isolated from
an SDS-PAGE gel and analyzed using MALDI. Both bands were identified as the
same glycoprotein. We are currently in the process of testing this compound to
confirm its DC-SIGN binding and inhibitory properties.
Conclusions:
We have identified a specific glycoprotein in human milk that binds to
DC-SIGN and can inhibit HIV-1 transfer to CD4+ T lymphocytes.
Identification of this molecule may provide for a better understanding of
mother-to-child transmission of HIV-1 via breastfeeding and provide a further
target for future microbicide development.
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