Apolipoprotein B (apo B) is the major protein species of LDL, but the elucidation of its structure has proven to be a most difficult task mainly because of its total insolubility in a delipidated form which may be related to high oxidation sensitivity.1 While widely varying molecular weights for apo B have been reported in recent years, it is now thought to have a molecular weight in excess of 200.000.2 ' 3 The LDL receptor present on the surface of mammalian cells binds certain plasma lipoproteins by recognizing sites which are present on apo B as well as on apo E. 4 ' 5 The binding of LDL to the LDL receptor initiates a series of events which has been called the LDL pathway.6 ' 7 The LDL is taken into the cell where it is hydrolyzed in lysosomes. The released cholesterol inhibits endogenous cholesterol synthesis at the level of the rate-limiting enzyme, hydroxymethyl glu- taryl coenzyme A reductase (HMG CoA reductase), stimulates cholesterol esterification, and inhibits synthesis of the LDL receptor itself.Monoclonal antibodies have proven useful in the study of protein structure and in the identification of functional domain of proteins. With the aid of monoclonal antibodies against human LDL, we are trying to identify and eventually purify and characterize the regions of apo B recognized by the LDL receptor. Here we describe seven monoclonal antibodies against LDL which are differentiated on the basis of their cross-reactivities with chemically modified LDL and their abilities to interfere with the LDL pathway. In addition, we describe a novel adaptation of the cotitration method 8 which allows a tentative mapping of the antigenic determinants.
Methods
Preparation of LDLBlood from normal subjects was collected into EDTA and the red blood cells were removed by centrifugation. Lipoprotein subfractions were prepared in a Beckman L5-65 ultracentrifuge with a 50.2 Ti rotor (Beckman Instruments Incorporated, Spinco Division, Palo Alto, California). LDL were isolated by successive ultracentrifugations 9 at 4° between densities of 1.030-1.050 g/ml, dialyzed exhaustively against phosphate-buffered saline (PBS) containing 1 mM EDTA, sterilized by ultrafiltration and stored at 4°C.
Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.
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