Ziqiang Guan

Lipidomics reveals a remarkable diversity of lipids in human plasma

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patients blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

SRD5A3 Is Required for Converting Polyprenol to Dolichol and Is Mutated in a Congenital Glycosylation Disorder

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

A Mouse Macrophage Lipidome

We report the lipidomic response of the murine macrophage RAW cell line to Kdo2-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 Å of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1s control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.

Subcellular organelle lipidomics in TLR-4-activated macrophages

Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls(-) bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H(+)-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.

Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates

Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5–15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ∆clsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ∆clsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.

Attenuated virulence of a Francisella mutant lacking the lipid A 4′-phosphatase

Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4′-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4′-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4′-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 106 wild-type but not 108 mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia.

193 nm photodissociation of larger multiply-charged biomolecules

Abstract In contrast to most ion dissociation methods, 193 nm ultraviolet photodissociation of electrosprayed melittin (2.8 kDa) and ubiquitin (8.6 kDa) molecular ions yields new c and z ions (backbone amine bond dissociation) that provide additional sequence information. Dissociation by collisions or infrared photons produce b and y ions; for cleavages between the same amino acids the c ion represents the addition of NH 2 to the b ion, and z the loss of NH 2 from the y ion, so that these ions can be differentiated by this ± 16.02 Da difference. However, 193 nm photodissociation of 12–29 kDa ions as yet does not give collectable product ions, and that of the very stable y 18 2+ ion from ubiquitin only effects a side chain loss. 193 nm irradiation of negative ions of all-T 30-mer DNA appears to eject electrons; apparently this is the first observation of electron photodetachment from multiply-charged negative ions.

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH3SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH3SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfide-bonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by -SH alkylation.