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Peroxidasin uses hypohalous acid oxidants to catalyze the formation of sulfilimine bonds in basement membranes. 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.

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Matrix metalloproteinases (MMPs) are extracellular proteases that can cleave extracellular matrix and alter signaling pathways. They have been implicated in many disease states, but it has been difficult to understand the contribution of individual MMPs, as there are over 20 MMPs in vertebrates. The vertebrate MMPs have overlapping substrates, they exhibit genetic redundancy and compensation, and pharmacological inhibitors are non-specific. In contrast, there are only two MMP genes in Drosophila, DmMmp1 and DmMmp2, which makes Drosophila an attractive system to analyze the basis of MMP specificity. Previously, Drosophila MMPs have been categorized by their pericellular localization, as Mmp1 appeared to be secreted and Mmp2 appeared to be membrane-anchored, suggesting that protein localization was the critical distinction in this small MMP family. We report here that products of both genes are found at the cell surface and released into media. Additionally, we show that products of both genes contain GPI-anchors, and unexpectedly, that GPI-anchored MMPs promote cell adhesion when they are rendered inactive. Finally, by using new reagents and assays, we show that the two MMPs cleave different substrates, suggesting that this is the important distinction within this smallest MMP family.

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation.

Dysnatremias or abnormalities in plasma [Na ] are often termed disorders of water balance, an unclear physiologic concept often confused with changes in total fluid balance. However, most clinicians clearly recognize that hypertonic or hypotonic gains or losses alter plasma [Na ], while isotonic changes do not modify plasma [Na ]. This concept can be conceptualized as the electrolyte free water balance (EFWB), which defines the non-isotonic components of inputs and outputs to determine their effect on plasma [Na ]. EFWB is mathematically proportional to the rate of change in plasma [Na ] (dP /dt) and, therefore, is actively regulated to zero so that plasma [Na ] remains stable at its homeostatic set point. Dysnatremias are, therefore, disorders of EFWB and the relationship between EFWB and dP /dt provides a rationale for therapeutic strategies incorporating mass and volume balance. Herein, we leverage dP /dt as a desired rate of correction of plasma [Na ] to define a stepwise approach for the treatment of dysnatremias.

Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.

In hyperglycemia, hypertonicity results from solute (glucose) gain and loss of water in excess of sodium plus potassium through osmotic diuresis. Patients with stage 5 chronic kidney disease (CKD) and hyperglycemia have minimal or no osmotic diuresis; patients with preserved renal function and diabetic ketoacidosis (DKA) or hyperosmolar hyperglycemic state (HHS) have often large osmotic diuresis. Hypertonicity from glucose gain is reversed with normalization of serum glucose ([Glu]); hypertonicity due to osmotic diuresis requires infusion of hypotonic solutions. Prediction of the serum sodium after [Glu] normalization (the corrected [Na]) estimates the part of hypertonicity caused by osmotic diuresis. Theoretical methods calculating the corrected [Na] and clinical reports allowing its calculation were reviewed. Corrected [Na] was computed separately in reports of DKA, HHS and hyperglycemia in CKD stage 5. The theoretical prediction of [Na] increase by 1.6 mmol/L per 5.6 mmol/L decrease in [Glu] in most clinical settings, except in extreme hyperglycemia or profound hypervolemia, was supported by studies of hyperglycemia in CKD stage 5 treated only with insulin. Mean corrected [Na] was 139.0 mmol/L in 772 hyperglycemic episodes in CKD stage 5 patients. In patients with preserved renal function, mean corrected [Na] was within the eunatremic range (141.1 mmol/L) in 7,812 DKA cases, and in the range of severe hypernatremia (160.8 mmol/L) in 755 cases of HHS. However, in DKA corrected [Na] was in the hypernatremic range in several reports and rose during treatment with adverse neurological consequences in other reports. The corrected [Na], computed as [Na] increase by 1.6 mmol/L per 5.6 mmol/L decrease in [Glu], provides a reasonable estimate of the degree of hypertonicity due to losses of hypotonic fluids through osmotic diuresis at presentation of DKH or HHS and should guide the tonicity of replacement solutions. However, the corrected [Na] may change during treatment because of ongoing fluid losses and should be monitored during treatment.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Collagen-IV (Col-IV) scaffolds, a primordial basement membrane component, enabled animal multicellularity, evolution and adaptation. These scaffolds provide tensile strength and tether macromolecules, forming supramolecular complexes that interact with cell-surface receptors and influence cell-behavior. Triple-helical Col-IV protomers, composed of three α-chains, with a trimeric globular NC1-domain at the C-terminus, oligomerize forming a NC1-hexamer structure that connects adjoining protomers of Col-IV , Col-IV , and Col-IV scaffolds. Hexamer formation and stability are driven by the extracellular chloride concentration- \"chloride pressure\". Hexamer structure is reinforced by six sulfilimine bonds forming covalent crosslinks that weld together trimeric NC1-domains of adjoining protomers. We recently found evidence that sulfilimine bonds, independent of chloride, stabilize the quaternary structure of the Col-IV hexamer of the Col-IV scaffold. Here, we sought to determine whether this function also pertains to the Col-IV scaffold that occurs ubiquitously across the animal kingdom, and whether bromine, a cofactor of peroxidasin in bond formation, are evolutionary conserved. We found that sulfilimine bonds stabilized the quaternary structure of the Col-IV hexamer of bovine, mouse and a basal cnidarian, , and that the mechanism of bond formation mediated by peroxidasin and bromide is evolutionary conserved. Analyses of the crystal structure of the NC1-hexamer revealed that sulfilimine bonds covalently fasten a clasp-motif across the trimer-trimer interface, interlocking the domain-swapping region of neighboring subunits, which reinforces the hexamer quaternary structure imposed by chloride conformational constraints. Collectively, our findings reveal that the sulfilimine-bond reinforcement is a critical event in Col-IV scaffold assembly enabling multicellularity, evolution and adaptation of metazoans, beginning with ancient cnidarians.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.