John H. Fessler

Peroxidasin: a novel enzyme-matrix protein of Drosophila development.

Peroxidasin is a novel protein combining peroxidase and extracellular matrix motifs. Hemocytes differentiate early from head mesoderm, make peroxidasin and later phagocytose apoptotic cells. As hemocytes spread throughout the embryo, they synthesize extracellular matrix and peroxidasin, incorporating it into completed basement membranes. Cultured cells secrete peroxidasin; it occurs in larvae and adults. Each 1512 residue chain of the three‐armed, disulfide‐linked homotrimer combines a peroxidase domain with six leucine‐rich regions, four Ig loops, a thrombospondin/procollagen homology and an amphipathic alpha‐helix. The peroxidase domain is homologous with human myeloperoxidase and eosinophil peroxidase. This heme protein catalyzes H2O2‐driven radioiodinations, oxidations and formation of dityrosine. We propose that peroxidasin functions uniquely in extracellular matrix consolidation, phagocytosis and defense.

A role for integrin in the formation of sarcomeric cytoarchitecture

We propose that integrins help to coordinate the differentiation of the internal, sarcomeric cytoarchitecture of a muscle fiber with its immediate environment and are essential for correct integration of muscle cells into tissue. We found that integrin alpha PS2 beta PS accumulated at contact regions of Drosophila embryo cells cultured in D-22 medium on Drosophila laminin. Myotubes formed, but subsequent addition of serum or fibronectin was needed for sarcomere formation: integrin and actin became concentrated at Z-bands; myosin and actin occurred between the Z-bands. This change failed to occur in the multinucleate myotubes derived from integrin beta PS null myospheroid mutants. In normal embryos/early larvae, integrin was located at Z-bands and at muscle insertions. Myogenesis and Z-bands were defective in myospheroid embryos. Attachment, spreading, and growth of myoblasts and neurons on the laminin substrate utilized different binding proteins and were independent of integrin.

Laminin A chain: expression during Drosophila development and genomic sequence.

A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N‐ and C‐terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information provided by in situ hybridization. Homologs of the G‐subdomains of this A chain, which occur in interacting regions of agrin, perlecan, laminin and sex steroid binding protein, may be involved in protein associations.

Glutactin, a novel Drosophila basement membrane-related glycoprotein with sequence similarity to serine esterases

Glutactin, a new acidic sulfated glycoprotein, was isolated from Drosophila Kc cell culture media. Immunofluorescence microscopy located it to embryonic basement membranes, particularly to the sequentially invaginated envelope of the central nervous system, muscle apodemes and dorsal median cell processes. Its chromosome locus is 29D. The nucleic acid sequence coding for the 1023 residue long polypeptide contains one intron and was confirmed by partial amino acid sequencing. Glutactin has a signal peptide and an amino domain of greater than 500 residues that strongly resembles acetylcholine esterases and other serine esterases, but lacks the catalytically critical serine residue. The amino and carboxyl domains of glutactin are separated by 13 contiguous threonine residues. Glutamine and glutamic acid make up 44% of glutactins very acidic carboxyl domain. Glutactin preferentially binds Ca2+ in the presence of excess Mg2+ and four of its tyrosines are O‐sulfated. Several similarities with mammalian entactin caused our previous, preliminary mention of glutactin as a putative Drosophila entactin, but sequence comparison now shows them to be different proteins.

Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins.

A Drosophila UDP‐glucose:glycoprotein glucosyltransferase was isolated, cloned and characterized. Its 1548 amino acid sequence begins with a signal peptide, lacks any putative transmembrane domains and terminates in a potential endoplasmic reticulum retrieval signal, HGEL. The soluble, 170 kDa glycoprotein occurs throughout Drosophila embryos, in microsomes of highly secretory Drosophila Kc cells and in small amounts in cell culture media. The isolated enzyme transfers [14C]glucose from UDP‐[14C]Glc to several purified extracellular matrix glycoproteins (laminin, peroxidasin and glutactin) made by these cells, and to bovine thyroglobulin. These proteins must be denatured to accept glucose, which is bound at endoglycosidase H‐sensitive sites. The unusual ability to discriminate between malfolded and native glycoproteins is shared by the rat liver homologue, previously described by A.J.Parodi and coworkers. The amino acid sequence presented differs from most glycosyltransferases. There is weak, though significant, similarity with a few bacterial lipopolysaccharide glycotransferases and a yeast protein Kre5p. In contrast, the 56‐68% amino acid identities with partial sequences from genome projects of Caenorhabditis elegans, rice and Arabidopsis suggest widespread homologues of the enzyme. This glucosyltransferase fits previously proposed hypotheses for an endoplasmic reticular sensor of the state of folding of newly made glycoproteins.

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

Microenvironmental Regulation by Fibrillin-1

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.

Alternative splicing of papilin and the diversity of Drosophila extracellular matrix during embryonic morphogenesis

Papilins are extracellular matrix proteins that share a particular, common order of types of protein domains. They occur widely, from nematodes to man, and can differ in the number of repeats of a given type of domain. Protein variety is increased by differential splicing of pre‐mRNA. We report that Drosophila, which has a compact genome, expresses three splice variants of papilin during embryogenesis in developmentally defined patterns. These isoforms have different numbers of Kunitz and IgC2 domains. The papilin isoforms are expressed in specific cell types and contribute to different extracellular matrices in gastrulation folds, early mesoderm, heart formation, basement membranes, and elaboration of the excorporeal peritrophic membrane that lines the gut. This finding indicates an unexpectedly broad spectrum of different pericellular matrices in Drosophila embryos. Such papilin‐containing matrices have developmental as well as functional significance, as we previously showed that both suppression of papilin synthesis and ectopic overexpression lethally disrupt organogenesis. Developmental Dynamics 226:634–642, 2003.

Conserved Neuron Promoting Activity in Drosophila and Vertebrate Laminin α1

Drosophila S2 cells were transfected with constructs that code for two portions of the Drosophila laminin α chain. Construct recαL coded for domains III, I/II, and G of laminin α. Construct recαS coded for only the COOH-most 12% of the I/II domain and the G domain. The corresponding polypeptides were isolated and characterized from the culture media. The recαL chain partly formed disulfide-linked heterotrimers with the endogenously produced β and γ laminin chains. Like normal Drosophila laminin, a substrate coating of either recαL or recαS supported neuron differentiation and neurite extension of primary Drosophila embryo cell cultures. However, at the same low concentrations, only Drosophila laminin-1, but neither recαL nor recαS supported myogenesis in these cultures. Previously, an overlapping set of dodecapeptides that covered a region of the murine laminin α1 chain similar to recαS had been synthesized and tested for cell culture support properties (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). The Drosophila laminin α homologues of the six most active vertebrate dodecapeptides were now synthesized and tested as substrates for differentiation of primary Drosophila embryo cells. Peptides that contained either the Drosophila sequence SIKVGV or the murine homologue, SIKVAV, provided support for neurite extension.