1H, 13C and 15N chemical shift assignment of the honeybee odorant-binding protein ASP2.

In insects, the transport of airborne, hydrophobic odorants and pheromones
through the sensillum lymph is generally thought to be accomplished by
odorant-binding proteins (OBPs). We report the structural and functional
properties of a honeybee OBP called ASP2, heterologously expressed by the yeast
Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis
followed by ion-spray mass spectrometry combined with microsequencing. The
pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be
identical to that of Bombyx mori OBP, suggesting that this pattern occurs
commonly throughout the highly divergent insect OBPs. CD measurements revealed
that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but
different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed
that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or
addition of a chaotropic agent. A general volatile-odorant binding assay
allowed us to examine the uptake of some odorants and pheromones by ASP2.
Recombinant ASP2 bound all tested molecules, except beta-ionone, which could
not interact with it at all. The affinity constants of ASP2 for these ligands,
determined at neutral pH by isothermal titration calorimetry, are in the
micromolar range, as observed for vertebrate OBP. These results suggest that
odorants occupy three binding sites per dimer, probably one in the core of each
monomer and another whose location and biological role are questionable. At
acidic pH, no binding was observed, in correlation with monomerization and a
local conformational change supported by CD experiments.

Briand L, Nespoulous C, Huet JC, Takahashi M, Pernollet JC.
Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding
protein from honeybee (Apis mellifera L.).
Eur J Biochem. 2001 Feb;268(3):752-60.

Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.).

A honeybee putative general odorant-binding protein ASP2 has been expressed in
the methylotrophic yeast Pichia pastoris. It was secreted into the buffered
minimal medium using either the alpha-factor preprosequence with and without
the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native
signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence
with the spacer peptide showed N-terminal heterogeneity, the recombinant
protein using the two other secretion peptides was correctly processed. Mass
spectrometry showed that the protein secreted using the natural peptide
sequence had a mass of 13,695.1 Da, in perfect agreement with the measured
molecular mass of the native protein. These data showed a native-like
processing and the three disulfide bridges formation confirmed by sulfhydryl
titration analysis. After dialysis, the recombinant protein was purified by
one-step anion-exchange chromatography in a highly pure form. The final
expression yield after 7-day fermentation was approximately 150 mg/liter. To
our knowledge, this is the first report of the use of a natural insect leader
sequence for secretion with correct processing in P. pastoris. The
overproduction of recombinant ASP2 should allow ligand binding and mutational
analysis to understand the relationships between structure and biological
function of the protein.

Briand L, Perez V, Huet JC, Danty E, Masson C, Pernollet JC.
Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris.
Protein Expr Purif. 1999 Apr;15(3):362-9.

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins
thought to participate in perireceptor events of odor-pheromone detection by
carrying, deactivating, and/or selecting odor stimuli. The honeybee queen
pheromone is known to play a crucial role in colony organization, in addition
to drone sex attraction. We identified, for the first time in a social insect,
a binding protein called antennal-specific protein 1 (ASP1), which binds at
least one of the major queen pheromone components. ASP1 was characterized by
cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ
hybridization showed that it is specifically expressed in the auxiliary cell
layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a
divergent member of the insect OBP family. The recombinant protein presented
the exact characteristics of the native protein, as shown by mass spectrometry,
and N-terminal sequencing and exclusion-diffusion chromatography showed that
recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major
components, opposite to another putative honeybee OBP, called ASP2. ASP1
biosynthetic accumulation, followed by nondenaturing electrophoresis during
development, starts at day 1 before emergence, in concomitance with the
functional maturation of olfactory neurons. The isobar ASP1b isoform appears
simultaneously to ASP1a in workers, but only at approximately 2 weeks after
emergence in drones. Comparison of in vivo and heterologous expressions
suggests that the difference between ASP1 isoforms might be because of
dimerization, which might play a physiological role in relation with mate
attraction.

Danty E, Briand L, Michard-Vanhee C, Perez V, Arnold G, Gaudemer O, Huet D, Huet
JC, Ouali C, Masson C, Pernollet JC.
Cloning and expression of a queen pheromone-binding protein in the honeybee: an
olfactory-specific, developmentally regulated protein.
J Neurosci. 1999 Sep 1;19(17):7468-75.

The Journal of neuroscience : the official journal of the Society for Neuroscience
Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.

Solution Structure Of The Honey Bee General Odorant Binding Protein Asp2 In Complex With Trimethylsilyl-D4 Propionate

putative odorant-binding protein ASP2 [Apis mellifera]

odorant binding protein ASP2 [Apis mellifera]

odorant binding protein ASP2 [Apis cerana cerana]

The host-vector system used was extremely efficient (around 100mg/L), see ref_2.

1H, 13C and 15N Chemical Shift Assignment of the Honeybee Odorant-binding
Protein ASP2

The 1H-15N NOESY and 1H-15N TOCSY experiments were collected on the 15N protein
in H2O (sample 15N_H20).
The HNCA,HN(CO)CA, HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH experiments were
collected on the 13C-15N protein in 100%H20 (sample 15N13C_H2O).
The HCCH-TOCSY and 13C and NOESY-HSQC experiments were collected on the 13C-15N
protein in 100% D2O (sample 13C15N_D2O).

Spectrometer equipped with a 5 mm TXI triple resonance, triple-axis gradient
probe.

automatic NOE assignment using ARIA combined with CNS