Brief Reports

Localization of the Human Phospholipid Transfer Protein Gene to Chromosome 20q12-q13.1

The understanding of the physiological and pathophysiological role of PLTP has greatly increased since the discovery of PLTP more than a quarter of century ago. A comprehensive review of PLTP is presented on the following topics: PLTP gene organization and structure; PLTP transfer properties; different forms of PLTP; characteristics of plasma PLTP complexes; relationship of plasma PLTP activity, mass and specific activity with lipoprotein and metabolic factors; role of PLTP in lipoprotein metabolism; PLTP and reverse cholesterol transport; insights from studies of PLTP variants; insights of PLTP from animal studies; PLTP and atherosclerosis; PLTP and signal transduction; PLTP in the brain; and PLTP in human disease.

PLTP's central role in lipoprotein metabolism and lipid transport in the vascular compartment has been firmly established. However, more studies are needed to further delineate PLTP's functions in specific tissues, such as the lung, brain and adipose tissue. Furthermore, the specific role that PLTP plays in human diseases, such as atherosclerosis, cancer, or neurodegenerative disease, remains to be clarified. Exciting directions for future research include evaluation of PLTP's physiological relevance in intracellular lipid metabolism and signal transduction, which undoubtedly will advance our knowledge of PLTP functions in health and disease. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).

Plasma phospholipid transfer protein (PLTP) is one of the key proteins in lipid and lipoprotein metabolism. We examined PLTP distribution in human brain using PLTP mRNA dot-blot, Northern blot, immunohistochemistry (IHC), Western blot, and phospholipid transfer activity assay analyses. PLTP mRNA of 1.8 kb was widely distributed in all the examined regions of the central nervous system at either comparable or slightly lower levels than in the other major organs, depending on the region. Cerebrospinal fluid phospholipid transfer activity represented 15% of the plasma activity, indicating active PLTP synthesis in the brain. Western blot and phosholipid transfer activity assay demonstrated secretion of active PLTP by neurons, microglia, and astrocytes in culture. IHC demonstrated PLTP presence in neurons, astrocytes, microglia, and oligodendroglia. Some neuronal groups, such as nucleus hypoglossus and CA2 neurons in hippocampus, ependymal layer, and choroid plexus were particularly strongly stained, with substantial glial and neuropil immunostaining throughout the brain.

Comparison between brain tissues from patients with Alzheimer's disease (AD) and nonAD subjects revealed a significant increase (P = 0.02) in PLTP levels in brain tissue homogenates and increased PLTP immunostaining in AD.

Phospholipid transfer represents (PLTP) the activity by which the protein is initially identified. PLTP can transfer all the common types of phosphatidylcholine as well as free cholesterol. The assay of PLTP activity in plasma samples is usually performed by measuring the transfer of radiolabeled phospholipids between liposomes and HDL. Mutation analysis revealed that the N-terminal lipid binding pocket predicted by computer modeling of the protein is essential for the phospholipid transfer activity. PLTP is also an HDL conversion factor. Based on the involvement of PLTP in preβ-HDL formation, it has been proposed that PLTP has a physiological function in the reverse cholesterol transport pathway, by which cholesterol is transported from extrahepatic tissues to the liver for excretion. The chapter further explains human conditions with elevated plasma PLTP activity levels. Plasma PLTP activity is elevated in a variety of disease states with increased risk for atherosclerosis and coronary heart disease, for example, diabetes mellitus type 1 and type 2.

The expression level of the neuronal-specific K–Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl⁻-dependent ⁸⁶Rb⁺ uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K–Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were performed in both isotonic and hypotonic conditions, revealing K_ms for K⁺ and Cl⁻ of 9.3±1.8 mM and 6.8±0.9 mM, respectively; both affinities are significantly higher than KCC1 and KCC4. The K_m for Cl⁻ is close to the intracellular Cl⁻ activity of mature neurons, as befits a neuronal efflux mechanism.

The phospholipid transfer protein (PLTP) shows a wide variety of functions including transfer of phospholipids and other lipid-like substances. Performing gene hunting in brain of patients with Down syndrome (DS) we detected the absence of a fragment identified as PLTP. Cerebellum of 4 controls, 7 patients with DS, 5 patients with Alzheimer's disease (AD) were used for differential display and for quantification of mRNA steady state levels of the isomer PLTP-1 by blotting methods. Differential display showed the absence of a cDNA fragment and cloning, sequencing and gene bank work revealed 100% homology with human PAC 337018 on chromosome 20q containing the PLTP gene. The PLTP gene in turn consists of at least three different PLTP isomers. Based on these results, a 450 bp cDNA fragment of the PLTP-isomer I (PLTP I) was isolated and amplified by PCR, serving as probe for the PLTP-1 isomer and its expression level was found to be significantly reduced in cerebellum of patients with DS. Biologically, the downregulation of PLTP maybe involved in the pathology of DS as phospholipids not only are of importance for membrane biogenesis and structure but also in the regulation of cellular metabolism, signaling and growth. In the brain, phospholipids in addition are integral constituents of myelins and synaptosomes (Johnson etc) and deficient PLTP levels may account for the deteriorated functions described to occur in DS brain.

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, α-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.