Show simple item record

dc.contributor.advisorChung, Nancy P.Y.
dc.contributor.advisorKewalRamani, Vineet N.
dc.contributor.authorKaramchandani, Jaideep M.
dc.date.accessioned2011-12-05T16:14:05Z
dc.date.available2011-12-05T16:14:05Z
dc.date.issued2009
dc.identifier.urihttp://hdl.handle.net/10920/24203
dc.descriptionix, 44 p.en_US
dc.description.abstractDendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) is a C-type lectin present in activated B-cells, subsets of macrophages, mucosal dendritic cells, and monocyte-derived dendritic cells. DC-SIGN has been proposed to be exploited as an attachment receptor for a wide variety of viral pathogens in cis and trans infections. It has been observed that human immunodeficiency virus 1 (HIV-l) is captured by activated B-cells, transformed B-cells (such as Raji, NC- 37, and Ramos), and dendritic cells expressing DC-SIGN and is efficiently transmitted to CD4+ T cells upon co-culture. In contrast to DC-SIGN, liverllymph node specific ICAM- 3-grabbing non-integrin (L-SIGN), a DC-SIGN-related molecule, which shares 77% homology to DC-SIGN, is less efficient in promoting HIV -1 transmission when expressed in B-cells. In this study, we sought to understand the structural differences between DC-SIGN and L-SIGN that influence HIV-I binding and transmission efficiency. Using a FACS-based binding assay with virus-like particles (VLPs) displaying HIV-l Envelope (Bnv), we observed that VLPs interacted with DC-SIGN but not L-SIGN. To map the domains within DC-SIGN that is necessary for virus binding, Raji lines expressing DC-SIGNIL-SIGN chimeras and point mutants were created by overlap PCR. Wild-type chimera and point mutant lectin expression levels were measured using lectin specific monoclonal antibodies. In addition, the ability of wild-type molecules, chimeras and point mutants to bind ICAM-3 was tested. Examination of the chimeras revealed that replacement of the DC-SIGN carbohydrate recognition domain (CRD) with the L-SIGN CRD was sufficient to impair VLP binding to background levels observed in Raji cell controls. By contrast, VLP binding was restored in L-SIGN chimeras containing the DC-SIGN CRD suggesting that the DC-SIGN CRD was necessary and sufficient for HIV -1 binding. Analysis of additional chimeric molecules indicated that DC-SIGN residues 253 to 288 were essential for efficient HIV -1 VLP binding. Point mutagenesis through reciprocal substitution and alanine scanning mutagenesis demonstrated that Tryptophan 258 (Trp258) and Glycine (Gly288) in alpha helical (a 1) region of individual monomers were specifically required for VLP binding. Based on the DC-SIGN crystal structure, these residues are proposed to contribute to alpha helical dimer interface stability within the CRD. Understanding the role of alpha helical dimer interface stability on HIV -1 and other virus pathogen binding may guide the development of antiviral therapies targeting the dimer interface.en_US
dc.format.mimetypeapplication/pdf
dc.language.isoen_USen_US
dc.publisherKalamazoo Collegeen_US
dc.relation.ispartofKalamazoo College Biology Senior Individualized Projects Collection
dc.relation.ispartofseriesSenior Individualized Projects. Biology;
dc.rightsU.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.
dc.titleDC-SIGN Carbohydrate Recognition Domain (CRD) Determinants Responsible for HIV -1 Bindingen_US
dc.typeThesisen_US
KCollege.Access.ContactIf you are not a current Kalamazoo College student, faculty, or staff member, email dspace@kzoo.edu to request access to this thesis.


Files in this item

Thumbnail

This item appears in the following Collection(s)

  • Biology Senior Individualized Projects [1489]
    This collection includes Senior Individualized Projects (SIP's) completed in the Biology Department. Abstracts are generally available to the public, but PDF files are available only to current Kalamazoo College students, faculty, and staff.

Show simple item record