Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

Proteolytic Processing of the Extracellular Scaffolding Protein LEV-9 Is Required for Clustering Acetylcholine Receptors

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.     

Identification of a Novel Lipofuscin Pigment (iisoA2E) in Retina and Its Effects in the Retinal Pigment Epithelial Cells

Lipofuscin accumulation in retinal pigment epithelial (RPE) cells of the eye implicates the etiologies of Stargardt disease and age-related macular degeneration, a leading cause of blindness in the elderly. Here, we have identified a previously unknown RPE lipofuscin component. By one- and two-dimensional NMR techniques and mass spectrometry, we confirmed that this compound is a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side chains are attached to adjacent carbons of a pyridinium ring. This pigment is a light-induced isomer of isoA2E, rather than A2E, referred to as iisoA2E. This pigment is a fluorescent lipofuscin compound with absorbance maxima at ∼430 and 352 nm detected in human, pig, mouse, and bovine eyes. Formation of iisoA2E was found in reaction mixtures of all-trans-retinal and ethanolamine. Excess intracellular accumulation of this adduct in RPE cells in vitro leads to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of the A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as biosynthetic precursors of isoA2E and iisoA2E.     

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro⁹⁸⁶ in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro⁹⁴⁴ in α1(II) and α2(V) and Pro⁷⁰⁷ in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro⁴⁷⁰ in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian α1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.     

Kindlin-2 Tyrosine Phosphorylation and Interaction with Src Serve as a Regulatable Switch in the Integrin Outside-in Signaling Circuit

Integrin-mediated cell-extracellular matrix (ECM) adhesion is critical for control of intracellular signaling; however, the mechanisms underlying this “outside-in” signaling are incompletely understood. Here we show that depletion of kindlin-2 impairs integrin outside-in signaling. Kindlin-2 is tyrosine-phosphorylated upon cell-ECM adhesion. Furthermore, kindlin-2 binds Src in a cell-ECM adhesion-regulatable fashion. At the molecular level, the kindlin-2·Src interaction is mediated by the kindlin-2 F0 and the Src SH2 and SH3 domains. Src activation increases kindlin-2 tyrosine phosphorylation and the kindlin-2·Src interaction. Conversely, inhibition of Src reduces kindlin-2 tyrosine phosphorylation and diminishes the kindlin-2·Src interaction. Finally, disruption of the kindlin-2·Src interaction, unlike depletion of kindlin-2, impairs neither cell-ECM adhesion nor cell-ECM adhesion-induced focal adhesion kinase Tyr-397 phosphorylation. However, it markedly inhibits cell-ECM adhesion-induced paxillin tyrosine phosphorylation, cell migration, and proliferation. These results suggest that kindlin-2 tyrosine phosphorylation and interaction with Src serve as a regulatable switch downstream of focal adhesion kinase in the integrin outside-in signaling circuit, relaying signals from cell-ECM adhesion to paxillin that control cell migration and proliferation.     

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition,Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6^−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6^−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6^−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.     

Cyr61, a Matricellular Protein,Is Needed for Dendritic Arborization of Hippocampal Neurons

The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a β1-integrin-dependent manner.     

Lateral Growth Limitation of Corneal Fibrils and Their Lamellar Stacking Depend on Covalent Collagen Cross-linking by Transglutaminase-2 and Lysyl Oxidases,Respectively

Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ∼35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.