Structural and functional characterization of human NAD kinase.

NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein.

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

Insect immune activation by recombinant Galleria mellonella apolipophorin III(1).

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.