The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.

ABSTRACT

The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2nd instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2nd instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2nd instar.