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The Proprotein Convertase Encoded by amontillado (amon) Is Required in Drosophila Corpora Cardiaca Endocrine Cells Producing the Glucose Regulatory Hormone AKH

Figure 5

Direct peptide profiling of AKH and AKHGK in control and amonC241Y flies.

(A) Model of the processing of the AKH prepropeptide (top) and profiling of the larval ring gland (left) and adult corpora cardiaca (right). AKH is processed by a concerted action of a signal peptidase (SP) and amon, likely followed by a two-step carboxypeptidase (CP) action that first removes the C-terminal R yielding the intermediate AKHGK. AKHG is than amidated to bioactive AKH (not shown). While AKH and AKHGK were detected in most preparations from control and rescued (continued heatshock once a day) flies, they were not detectable in amon larvae. (B) Original direct mass profiles from corpora cardiaca of adult control (above) and amon (below) flies. AKH only occurs as the characteristic [M+Na]+ and [M+K]+ adducts, whereas AKHGK also occurs as [M+H]+. In the control fly, both peptides show higher signal intensities as the stable isotope-labelled standard peptide (AKH*). In the amon fly, the signal intensity is clearly higher for AKH* than for the native peptides. As previously reported [41], [42], no other mass peaks occur in the range 990-1220 Da in direct mass spectrometric CC profiles. (C) Standard curve for adult corpora cardiaca obtained with a dilution series of AKH* added to the matrix salt, male OrR wild-type flies. The y axis shows the signal intensity ratio of native AKH/AKH*. Error bars are S.E.M. The relationship of AKH/AKH* is linear for AKH* concentrations of 50–500 nM. (D) Peptide quantification with the labeled AKH* standard at 400 nM. The concentrations of both AKH and AKHGK are significantly reduced in amon flies vs. controls five days after eclosion and final heatshock. *p<0.05, **p<0.01, Mann-Whitney.

Figure 5