Sulfatide: a multifunctional glycolipid constituent of biological membranes

Sulfogalactosylceramide (or sulfatide) has been localized in the central nervous system by indirect immunofluorescence. This glycosphingolipid belongs essentially to the myelinated areas. Nevertheless, it has also been found in ependymal cells, subpial processes and, in the cerebellum, in the Bergmann fibers. In the brain areas, known to be enriched in opiate receptors, some cells and nerve terminals are also sulfatide positive. In this latter localization, opiates were shown to selectively inhibit binding of the antisulfatide antibodies. We have also shown that antisulfatide inhibited, in vitro, the stereo-specific binding of narcotic drugs and that they antagonized the in vivo effects of morphine and beta-endorphin.     

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

The plasma membrane as an adaptable fluid mosaic

The fluid mosaic model proposed by Singer and Nicolson established a powerful framework to interrogate biological membranes that has stood the test of time. They proposed that the membrane is a simple fluid, meaning that proteins and lipids are randomly distributed over distances larger than those dictated by direct interactions. Here we present an update to this model that describes a spatially adaptable fluid membrane capable of tuning local composition in response to forces originating outside the membrane plane. This revision is rooted in the thermodynamics of lipid mixtures, draws from recent experimental results, and suggests new modes of membrane function.     

Functional patchworking at the plasma membrane

Lipids and proteins are not evenly distributed within the plasma membrane (PM), but instead segregate laterally into many specialized microdomains whose functional relevance is not clear. In this issue, Busto et al ( 2018 ) demonstrate that substrate flux through a nutrient transporter drives the lateral relocation of the transporter between specific microdomains at the yeast PM, suggesting that regulating the lateral plasma membrane compartmentalization for individual proteins could be a general process for cellular response to environmental conditions.     

The prostate plasma membrane as an androgen receptor.

The pivotal role of the cell nucleus in androgenic control of target organs, such as the prostate, has become increasingly suspect. Equally qualified receptor activities have been found in the cytosol, endoplasmic reticulum, and plasma membrane. It is presently difficult to explain how a sex steroid can manage proliferation, metabolism, biosynthesis and secretion, all through chromatin-directed signals. In my search for a more satisfactory mediator of androgen action, I discovered that the sodium-potassium-dependent ATPase of the prostate plasma membrane binds androgen, and is activated by the hormone's presence to serve as a metabolic pacemaker. This paper is my terminal status report on one aspect of this hypothesis; namely, the nature and site of androgen binding, with clues as to the mode of action. SDS-PAGE indicates that androgen can be bound to the beta-subunit of prostatic Na,K-ATPase. Selective enrichment of the enzyme by reversible coupling to either concanavalin A or a DHT-affinity column support this conclusion. Several studies show the dynamic effect of androgen binding: increased ouabain binding; enhancement of this binding by facilitated phosphorylation; spectroscopic evidence of conformational shifts, possibly consequences of these suggested activities for regulation, especially of metabolism, are examined.     

Functional roles of plasma membrane localized estrogen receptors

A series of emerging data supports the existence and importance of plasma membrane localized estrogen receptors in a variety of cells that are targets for the steroid hormone action. When estradiol (E2) binds to the cell surface protein, the ensuing signal transduction event triggers downstream signaling cascades that contribute to important biological functions. Aside from the classical signaling through nuclear estrogen receptors, we have provided evidence for the functional roles of an estrogen receptor localized in the plasma membrane. This review highlights some of the recent advances made in the understanding of the genomic/non-genomic actions of plasma membrane localized estrogen receptors.     

PKD2 functions as an epidermal growth factor-activated plasma membrane channel

PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK(1) kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP(2)) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) does not have any effect on this conductance. Overexpression of type Ialpha phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kalpha], which catalyzes the formation of PIP(2), suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-gamma2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP(2) in the primary cilium of LLC-PK(1) cells. We propose that plasma membrane PKD2 is under negative regulation by PIP(2). EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP(2)-mediated inhibition.     

Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.     

Functional implications of plasma membrane condensation for T cell activation

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.     

Synthesis of an O-sulfo Lewis analog as glycolipid antigen

The title compound containing dihydroceramide as a ligand for CD1d was accomplished using the mannosyl, glucosaminyl, and fucosyl donors, and a sphinganine analogue, as suitable building blocks. The 2-O-unprotected mannosyl donor was coupled effectively with the sphinganine analog to afford the mannnosyl sphinganine derivative. The coupling of the glucosaminyl donor with the mannnosyl sphinganine acceptor required triflic acid as a promoter and the promoter change to silver triflate led to the undesired glycal production. The reduction of azide group using Zn powder was the key process, in which the amount of acetic acid was restricted to avoid the benzoyl migration and N-trichloroacetyl deprotection. The trisaccharide glycolipid was sulfonated at the 3-position of fucose moiety.     

Bacterial 'histone-like protein I' (HLP-I) is an outer membrane constituent?

The nucleoid-associated 'histone-like protein I' (HLP-I) protein of E. coli was found to be homologous with the cationic 16-kDa outer membrane protein OmpH of Salmonella typhimurium. Deduced from the nucleotide sequence, the HLP-I protein has 91% identical residues with the OmpH protein. Both proteins have very similar cleavable signal sequences. The nucleotide sequence similarity between the corresponding genes hlpA and ompH is 87%. The ompH gene is located in a gene cluster resembling the hlpA-ORF17 region of E. coli which is close to the Ipx genes involved in the biosynthesis of lipopolysaccharides. The localization of the OmpH/HLP-I protein in the cell is discussed.     

Lanthionine as an essential constituent of cell wall peptidoglycan ofFusobacterium nucleatum

Lanthionine, a sulfur-containing diamino acid which had not previously been reported as one of the main amino acids of any bacterial cell wall peptidoglycan, was demonstrated inFusobacterium nucleatum peptidoglycan isolated by sodium dodecyl sulfate extraction and protease digestion. Lysine, diaminopimelic acid, and ornithine were absent. Lanthionine seems to be an essential dibasic amino acid, involved in cross-linkages betwen stem peptide subunits inF. nucleatum.