Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

The origins and developments of sulfation-prone tyrosine-rich and acidic N- and C-terminal extensions of class ll and lll small leucine-rich repeat proteins shed light on connective tissue evolution in vertebrates.

BACKGROUND: Small leucine-rich repeat protein (SLRP) family members contain conserved leucine-rich repeat motifs flanked by highly variable N- and C-terminal regions. Most class II and III SLRPs have tyrosine-rich N-terminal regions and some of these are sulfated. However, the evolutionary origin and conservation of the tyrosine-rich and acidic terminal regions remain undetermined. In this study, we present the most comprehensive multiple sequence alignment (MSA) analyses of all eight class II and III SLRPs to date. Based on the level of conservation of tyrosine residues and adjacent sequences, we predict which tyrosine residues are most likely to be sulfated in the terminal regions of human class II and III SLRPs. RESULTS: Using this novel approach, we predict a total of 22 tyrosine sulfation sites in human SLRPs, of which only 8 sites had been experimentally identified in mammals. Our analyses suggest that sulfation-prone, tyrosine-rich and acidic terminal regions of the class II and III SLRPs emerged via convergent evolution at different stages of vertebrate evolution, coinciding with significant evolutionary events including the development of endochondral bones and articular cartilage, the aquatic to terrestrial transition, and the formation of an amnion. CONCLUSIONS: Our study suggests that selective pressures due to changes in life conditions led to the formation of sulfotyrosine-rich and acidic terminal regions. We believe the independent emergence and evolution of sulfotyrosine-rich and acidic N- and C-terminal regions have provided each class II and III SLRP member with novel vital functions required to develop new specialized extracellular matrices and tissues in vertebrate species.

Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model.

The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was inhibited by L-glutamate and L-aspartate (1 mM). L-glutamate uptake followed Michaelis-Menten-like kinetics with high and low affinity at the abluminal and luminal membrane, respectively. This indicated that L-glutamate is taken up via EAAT-1 at the abluminal membrane and exits at the luminal membrane via a low affinity glutamate/aspartate transporter. Metabolism of L-glutamate and transport of metabolites was examined using [U-13C] L-glutamate. Intact L-glutamate and metabolites derived from oxidative metabolism were transported through the endothelial cells. High amounts of L-glutamate-derived lactate in the luminal medium indicated cataplerosis via malic enzyme. Thus, L-glutamate can be transported intact from brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites.

LASIK surgery of granular corneal dystrophy type 2 patients leads to accumulation and differential proteolytic processing of transforming growth factor beta-induced protein (TGFBIp).

More than 60 mutations in transforming growth factor beta-induced protein (TGFBIp) have been reported in humans causing a variety of phenotypic protein aggregates in the cornea, commonly termed corneal dystrophies. One mutation, generating an arginine to histidine amino acid substitution at position 124 in mature TGFBIp leads to granular corneal dystrophy type 2 (GCD2). Homozygous GCD2 cases develop massive protein accumulation early in life whereas heterozygous GCD2 cases become affected much later and generally with a much less severe outcome. However, if heterozygous GCD2 patients undergo laser-assisted in situ keratomileusis (LASIK) surgery protein accumulation is accelerated and they develop massive protein accumulations a few years after surgery. Here, we present the protein profile of aggregate-containing corneal tissue from GCD2 patients with a history of LASIK surgery using LC-MS/MS. Label-free quantification of corneal extracellular matrix proteins showed accumulation of TGFBIp. This was supported by 2DE and immunoblotting against TGFBIp that revealed the accumulation of full-length TGFBIp. In addition, a high molecular weight TGFBIp complex was more apparent in GCD2 patients after LASIK surgery, which may be important for the disease progression. Lastly, 2DE also revealed differential processing between GCD2 patients with a history of LASIK surgery when compared to healthy individuals.

Fibril Core of Transforming Growth Factor Beta-Induced Protein (TGFBIp) Facilitates Aggregation of Corneal TGFBIp.

Mutations in the transforming growth factor beta-induced (TGFBI) gene result in a group of hereditary diseases of the cornea that are collectively known as TGFBI corneal dystrophies. These mutations translate into amino acid substitutions mainly within the fourth fasciclin 1 domain (FAS1-4) of the transforming growth factor beta-induced protein (TGFBIp) and cause either amyloid or nonamyloid protein aggregates in the anterior and central parts of the cornea, depending on the mutation. The A546T substitution in TGFBIp causes lattice corneal dystrophy (LCD), which manifests as amyloid-type aggregates in the corneal stroma. We previously showed that the A546T substitution renders TGFBIp and the FAS1-4 domain thermodynamically less stable compared with the wild-type (WT) protein, and the mutant FAS1-4 is prone to amyloid formation in vitro. In the present study, we identified the core of A546T FAS1-4 amyloid fibrils. Significantly, we identified the Y571-R588 region of TGFBIp, which we previously found to be enriched in amyloid deposits in LCD patients. We further found that the Y571-R588 peptide seeded fibrillation of A546T FAS1-4, and, more importantly, we demonstrated that native TGFBIp aggregates in the presence of fibrils formed by the core peptide. Collectively, these data suggest an involvement of the Y571-R588 peptide in LCD pathophysiology.