Grant: Discovery Grants Program - Individual

Lipoprotein(a) (Lp(a)) is a fat/protein particle present in human blood. It is related to low density lipoprotein (LDL, also known as the ""bad cholesterol"") but contains an additional protein component termed apolipoprotein(a) (apo(a)). Elevated levels of Lp(a) are a risk factor for the development of cardiovascular diseases such as heart attacks and strokes. While it is known that the levels of Lp(a) are controlled by the rate of the biosynthesis of the particle (rather than its clearance from the bloodstream), the exact mechanisms of Lp(a) biosynthesis remain largely unknown or controversial. In particular, we wish to examine the site of Lp(a) assembly from LDL and apo(a), the exact nature of the lipoprotein particle that apo(a) assembles with, the identity and characteristics of the enzyme that catalyzes Lp(a) assembly, and the identity of the specific amino acid on the protein component of LDL (known as apoB100) that apo(a) links to. When Lp(a) is assembled, apo(a) first makes noncovalent (transient) contacts with apoB100, and then a specific covalent (permanent) bond is formed between the two proteins called a disulfide bond. We and others have reported that assembly of apo(a) and LDL takes place outside the cell, possibly on the cell surface. Using different methodology, others have indirectly shown that assembly may happen inside the cell. To resolve this controversy, we will look for complexes between apo(a) and apoB100 inside the cell that are either noncovalent or covalent. We will also compare the rates of secretion of apo(a) and apoB100 that is destined for either LDL or Lp(a). It has even been suggested that apo(a) assembles with a particle distinct from LDL, yet one that still contains apoB100. Therefore, we will measure if apo(a) is assembled more or less efficiently with apoB100-containing particles of different size and density. As part of these studies, we will determine if the size of apo(a) influences the rate of assembly; apo(a) can differ remarkably in size between different individuals and small apo(a) sizes are associated with higher Lp(a) levels. We have some preliminary evidence about the nature of the enzyme that catalyzes disulfide bond formation between apo(a) and apoB100, which we call Lp(a) oxidase, and we will attempt to either purify the oxidase or determine if one of several known oxidases can perform this reaction. Finally, we will directly identify the amino acid (cysteine) in apoB100 that forms a disulfide bond with apo(a) by cleaving Lp(a) into several large pieces, identifying the piece that contains both apo(a) and apoB100 segments (and thus the disulfide bond that joins them), and then characterizing the sequence of the segments using a technique called mass spectrometry. Our research will both address longstanding questions as well as break exciting new ground in our understanding what biochemical mechanisms underlie control of the biosynthesis of Lp(a).