Identification of a biomarker in cerebrospinal fluid for neuronopathic forms of Gaucher disease.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation.

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the SARAF-fold. The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the SARAF luminal switch that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation.

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the "SARAF-fold." The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the "SARAF luminal switch" that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation

Elevation of GPNMB levels in CSF and brain of nGD patients.

<p>(A) Levels of GPNMB determined by LC-MS/MS in CSF of four type 3 GD patients. Results are means ± SEM. ** <i>p</i>< 0.01. (B) Levels of GPNMB in CSF of four type 3 GD patients determined by ELISA. Results are means ± SEM (n = 4). ** <i>p</i><0.01. (C) Western blot of GPNMB in CSF of control and a type 3 GD patient (sample designation 4). Results are from a typical experiment repeated 3 times. (D) Levels of GPNMB in nGD brain determined by ELISA(n = 3 for control, n = 6 for nGD (type 2 and type 3 patients). Results are means ± SEM, ** <i>p</i>< 0.01</p

GPNMB peptides identified by LC-MS/MS.

<p>The GPNMB sequence is shown (UniProtKB/Swiss-Prot Q14956), with the site of cleavage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120194#pone.0120194.ref020" target="_blank">20</a>] indicated in red and the two peptides identified by LC-MS/MS indicated in green and brown.</p

GPNMB levels in brain and serum of Gba^flox/flox; nestin-Cre mice.

<p>Levels of GPNMB in (A) brain (n = 3) at different days post-natal (p) and (B) serum (n = 4,n = 5) of 21-day old Gba^flox/flox; nestin-Cre mice determined by ELISA. Results are means ± SEM *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Up-regulated proteins in the CSF of type 3 GD patients.

<p>CSF from type 3 GD patients and age matched controls (n = 4) was digested with trypsin and subjected to label-free quantitative global proteomic analysis using liquid chromatography and tandem mass spectrometry (LC-MS/MS).</p><p>Up-regulated proteins in the CSF of type 3 GD patients.</p