Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

Abstract

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å , which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.

Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

“…However, the two PEases, Demetra and Ceres, are an arylphorin and an hexamerin, respectively 51 , and they are phylogenetically related to phenoloxidases (enzymes targeting aromatic rings) and hemocyanins, oxygen transport proteins that also present phenoloxidase activity 52,53 . In the literature diverse functions are attributed to this family of proteins, such as storage 54 , immunity 55 , and defence against plant phenolics 56 . The presence of this type of enzymes in the saliva of insects, already described in the literature [57][58][59] , has a clear functional interpretation as the first line of defence against pathogens and plant anti-herbivore mechanisms [57][58][59] .…”

cDNA sequences of two arylphorin subunits of an insect biliprotein: phylogenetic differences and gene duplications during evolution of hexamerins-implications for hexamer formation

“…As the crystal structures of the homomeric arylphorin (APA) from A. pernyi and of two heteromeric arylphorins from B. mori (Ryu et al., ; Pietrzyk et al., ; Hou et al., ) are almost conserved, the overall structure of the heteromeric Cerura protein is thought to be comparable. This sound assumption provided the basis for the use of the APA structure to build a model of the hexameric apoprotein of CVA, presented in Figure , which is discussed below.…”

“…The view of the trimers as the building units of the hexamers has received strong support by the recent crystal structure analyses of two heteromeric arylphorins from silkworm hemolymph (Pietrzyk et al., ; Hou et al., ). One of these arylphorins is best described as a dimer of different homotrimers, while the other one represented a trimer of heterodimers.…”

“…This was first demonstrated by the crystal structure analysis of a recombinant homomeric Antheraea pernyi arylphorin (APA), the Chinese oak silkworm (Ryu et al., ). Recently, two independent studies of the crystal structures of heteromeric arylphorins from the hemolymph of the mulberry silkworm, Bombyx mori , confirmed the view of a conserved quaternary structure of these evolutionarily related hexamers (Pietrzyk et al., ; Hou et al., ).…”

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva

“…In particular, each monomer is glycosylated at two sites, one located in the cleft formed at the Cibeles-Demetra interface (Asn 211 in both proteins) and the other found in the external face of the monomer (Asn 481 in Cibeles and Asn 479 in Demetra), branching out from the Ig-like subdomain and making contact in cis with the N-terminal region of the same monomer (Fig 3, B and C). The presence of glycosylations has been described in other arylphorins, in which they are frequently involved in protein folding and stabilization (38)(39)(40).…”