Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1.
BACKGROUND
Anosmin-1, the protein implicated in the X-linked Kallmann's syndrome, plays a role in axon outgrowth and branching but also in epithelial morphogenesis. The molecular mechanism of its action is, however, widely unknown. Anosmin-1 is an extracellular protein which contains a cysteine-rich region, a whey acidic protein (WAP) domain homologous to some serine protease inhibitors, and four fibronectin-like type III (FnIII) repeats. Drosophila melanogaster Kal-1 (DmKal-1) has the same protein structure with minor differences, the most important of which is the presence of only two FnIII repeats and a C-terminal region showing a low similarity with the third and the fourth human FnIII repeats. We present a structure-function analysis of the different DmKal-1 domains, including a predicted heparan-sulfate binding site.


RESULTS
This study was performed overexpressing wild type DmKal-1 and a series of deletion and point mutation proteins in two different tissues: the cephalopharyngeal skeleton of the embryo and the wing disc. The overexpression of DmKal-1 in the cephalopharyngeal skeleton induced dosage-sensitive structural defects, and we used these phenotypes to perform a structure-function dissection of the protein domains. The reproduction of two deletions found in Kallmann's Syndrome patients determined a complete loss of function, whereas point mutations induced only minor alterations in the activity of the protein. Overexpression of the mutant proteins in the wing disc reveals that the functional relevance of the different DmKal-1 domains is dependent on the extracellular context.


CONCLUSIONS
We suggest that the role played by the various protein domains differs in different extracellular contexts. This might explain why the same mutation analyzed in different tissues or in different cell culture lines often gives opposite phenotypes. These analyses also suggest that the FnIII repeats have a main and specific role, while the WAP domain might have only a modulator role, strictly connected to that of the fibronectins.
Andrenacci
Davide
D
Institute of Genetics and Biophysics, A, Buzzati Traverso, CNR, 80131 Napoli, Italy. andrenacci@igb.cnr.it
Grimaldi
Maria R
MR
Panetta
Vittorio
V
Riano
Elena
E
Rugarli
Elena I
EI
Graziani
Franco
F
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019521:Body Patterning / Q000235:genetics / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000254:growth & development*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005353:Fibronectins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D006257:Head / Q000196:embryology / Q000254:growth & development; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009420:Nervous System / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014921:Wings, Animal / Q000254:growth & development
D029721:Drosophila Proteins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; C500363:Kal1 protein, Drosophila; D011993:Recombinant Fusion Proteins