Identification of an Amino-Terminal Fragment of Apolipoprotein E4 that Localizes to Neurofibrillary Tangles of the Alzheimer’s Disease Brain

Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer’s disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab consistently identified the predicted amino-terminal fragment (~18 kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18 kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain

Determination of Sulfated Glycosaminoglycan Binding Sites Within Collagen Type XI Using Surface Plasmon Resonance and Nuclear Magnetic Resonance Spectroscopy

Type XI Collagen is a minor constituent of the extracellular matrix of cartilage and is essential in the regulation of collagen fibril assembly and diameter. The alpha 1 chain of Collagen XI (Collagen a1(XI) ) contains a variable region that is modulated by alternative splicing in a tissue-specific and developmental manner. Preliminary data suggests that some Collagen XI isoforms interact with sulfated gylcosaminoglycans. Sulfated glycosaminoglycans are unbranched polysaccharides containing a disaccharide subunit. Chondroitin sulfate is a sulfated glycosaminoglycan which contains repeating disaccharide subunits composed of D-glucuronate and N-acetyl-D-glalactosamine sulfate. Chondroitin sulfate is located primarily in the extracellular matrix of cells. The function of chondroitin sulfate is not well understood, but has been shown to interact with proteins, and may play structural roles within the body. Biophysical methods will be used to assess the binding interactions between Collagen a1(XI) and the glycosaminoglycan chondroitin sulfate. Collagen a1(XI) isoform V1B was expressed in Escherichia coli and purified using affinity chromatography. Peptides pertaining to potential glycosaminoglycan binding sites in the variable region of the V1B isoform of Collagen a1(XI) were synthesized. Surface plasmon resonance spectroscopy was used to determine the binding interactions between Collagen a1(XI) peptides and chondroitin sulfate glycosaminoglycans. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of the V1B peptides and the physical interactions occuring between the peptides and chondroitin sulfate