Identification, characterization, and tissue distribution of apolipoprotein D in the rat

We recently described an unknown apolipoprotein that is present on the lipoprotein particles isolated from regenerating rat sciatic nerves. In the regenerating nerve, the concentration of this apolipoprotein rises 500-fold over its concentration in the normal nerve. In *is report we have identified the apolipoprotein by partial amino acid sequence analysis as apolipoprotein (apo) D. Characterization of rat apoD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of a series of molecular weight isoforms of between 27 kDa and 31 kDa that increase 2 kDa in apparent molecular mass upon reduction. Rat apoD has multiple isoelectric points between pH 4.05 and 4.37, apparently resulting from N-linked glycosylation. In the rat, unlike the human, little apoD is found in plasma. However, immunocytochemical localization of apoD in 12 tissues (liver, kidney, bladder, adrenal, cerebrum, duodenum, testis, lung, spleen, pancreas, heart, and skin) showed that a variety of cells contained substantial levels of apolipoprotein. llllD The broad distribution of apoD suggests that it may play a general role in cellular metabolism. Moreover, many of the same cell types varied dramatically in their content of apoD in different tissues, suggesting that the uptake or secretion of apoD by cells is regulated. Boyles, J. K., L. M. Notterpek, M. R. Wardell, and S. C. Rall, Jr. Identification, characterization, and tissue distribution of apolipoprotein D in the rat. J. Lipid Res. 1990. 31: 2243-2256. Supplementary key words vous system central nervous system Wallerian degeneration peripheral nerIn the regenerating peripheral nerve, massive membrane degradation and lipid release is rapidly followed by massive membrane biogenesis and lipid utilization. Peripheral nerve regeneration, therefore, serves as an excellent model in which to establish the mechanism of lipid transport between cells. After a denervating injury, as the nerve undergoes Wallerian degeneration, large quantities of phospholipids and cholesterol are released by the breakdown of myelin (1-5). Much of this lipid remains in the nerve, stored in macrophages, neurolemmal cells, and Schwann cells (2, 6-8). Large quantities of lipid are in turn required for the assembly of new axonal and myelin membranes. Both stored lipids and lipids produced locally or imported from the plasma are utilized in this process (2, 6-9). During nerve regeneration, lipids and lipidbinding proteins accumulate in lipoprotein particles within the interstitial matrix of the nerve (8, 10). Presumably, it is these lipoprotein particles that provide the nerve with a lipid transport system. It has been our goal to understand this lipid transport system. We and others have already identified three of the apolipoproteins that are components of the lipoprotein particles accumulating in the regenerating peripheral nerve of the rat: apolipoprotein (apo) E (8, lo), a p d I (8), and a@-IV (11). Among the several unidentified proteins is one that is produced by neurolemmal cells and increases in concentration over 500-fold during regeneration of the rat sciatic nerve (11). O n the first day after injury, this protein increases severalfold in concentration, and it reaches its peak concentration at 3-4 weeks after injury. Thus, both during axon regeneration (1 day to 2 weeks after injury) (8) and active myelination (2-6 weeks after injury) (8), this protein is present in greatly increased concentrations. We have now isolated, characterized, and identified this protein. It is apoD. Apolipoprotein D had previously been identified only in humans and the baboon, where it is a minor component of plasma lipoprotein particles (1216). The human apoD protein has been characterized (1216), and its mRNA (17) and gene (18) sequences have been determined. In the human, apoD mRNA has been identified in several tissues, including the brain (17, 19). Rat apoD, as shown by our study, is very similar to human apoD. Unlike the human protein, the rat protein is not Abbreviations: apo, apolipoprotein; HDL, high density lipoproteins; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; LCAT, 1ecithin:cholesterol acyltransferase. 'To whom correspondence should be addressed at: Gladstone Foundation Laboratories, P.O. Box 40608, San Francisco, CA 94140-0608. Journal of Lipid Besearch Volume 31, 199