| Abstract |
Scavenger receptor class B type I (SR-BI) is the functional receptor for high density
lipoprotein (HDL) and its three-dimensional structure has not been resolved. The main
objective of my thesis was to produce sufficient quantities of homogeneously modified
pure SR-BI suitable for crystallization studies. The long-term goal is to correlate the 3D
structure with the known functions of SR-BI in the selective uptake of lipids and the
efflux of free cholesterol. In order to produce large quantities of SR-BI with
homogeneous modifications, the receptor was expressed in a mutant Human Embryonic
Kidney (HEK) cell line, using a tetracycline-inducible system. The inducible expression
of SR-BI in cell cultures in a bioreactor overcame the problem of cell aggregation and
enhanced the protein yield. The mutant HEK cell line used for the expression of SR-BI
was mutated in the N-acetyl Glucosaminyl Transferase I gene (GnTI). This mutation led
to uniform glycosylation, with the high-mannose oligosaccharides GlcNAc(Man)5. The
glycosylation properties of SR-BI were verified by Endoglycosidase H (EndoH) and NGlycosidase
F (PNGase F) digestions. PNGase F digests high mannose, as well as
complex glycans. EndoH digests only high-mannose glycans. Western blotting analysis
following these digestions verified the production of SR-BI with the expected degree of
glycosylation (Μr ~54kDa). SR-BI produced on a small scale in cell cultures was purified
by immunoaffinity chromatography from the cell lysates using Sepharose coupled to the
monoclonal antibody 1D4. The antibody recognizes the epitope t1, a nonapeptide
(TETSQVAPA) that corresponds to nine C-aminoacids of rhodopsin. The t1 epitope was
engineered into the carboxy-terminus of SR-BI. Bound SR-BI was eluted by excess t1
and was detected by SDS-PAGE, Western blotting and silver staining. For large-scale
spinner cultures the mutant HEK line (HEK293SGnTI-TetR) was grown in a CELLIGEN
PLUS bioreactor, where the temperature and the pH value as well as the oxygen,
nitrogen, carbon dioxide and air content of the culture were controlled electronically. The
cell density achieved in this bioreactor was approximately 5 Χ 106 cells/ ml, total volume
5.5L). Using large-scale immunoffinity chromatography, 5-8 milligrams of pure protein
were produced per 5.5 liters of culture. Part of the purified protein was incorporated into
liposomes composed of cholesterol and phosphatidylcholine (and in some cases,
sphingomyelin) which were used for a filter binding assay. This assay allowed the
calculation of the quantity of lipoproteins bound to the proteoliposomes and of the
cholesteryl ether taken up by them via SR-BI. These analyses showed that the purified
glycosylation mutant SR-BI-t1 is functional and that it binds 125Ι-HDL and uptakes
cholesteryl ether from [3Η]-CE-HDL at levels similar to those of the wild type mSR-BIt1.
Increase in the concentration of CaCl2 in the buffer leads to increased binding of HDL,
but did not affect the uptake of cholesteryl ether. Binding of HDL and LDL is greatest at
37oC, while uptake of their cholesteryl ether increased as temperature increased. Binding
of HDL increased as pH decreased, without affecting the uptake of cholesteryl ether.
Addition of sphingomyelin in the liposomes increased the uptake of cholesteryl ether
from HDL, but did not affect the binding of 125I-HDL The chemical inhibitors BLT-1, 2,
3, 4 and 5 block the selective lipid uptake from HDL. These experiments revealed that
the target of the BLTs is SR-BI itself. Substitution of the thiol group of BLT-1, which
belongs to the thiosemicarbazone family of compounds, with an oxygen atom, created an
inactive isoform of BLT-1. These studies also revealed the importance of the
hydrophobic tail of BLT-1 for the inhibition of the SR-BI functions. A portion of the
protein isolated by immunoaffinity chromatography underwent further purification by
size exclusion and ion exchange chromatography, which showed that SR-BI preparations
were homogeneously pure. The effect of pH, dithiothreitol (DTT), temperature and the
age of the protein samples on their stability and oligomerization of SR-BI during the
chromatography were also assessed. Crystallization of the purified SR-BI generated small
hexagonal crystals 30-40 μm in length, which are being used currently in x-ray
crystallographic studies for the elucidation of the atomic structure of SR-BI.
|
Search
