LH mutant mice form similar MBL-A oligomers as wild type mice.

<p>(A) In serum of 2 months old female and male LH mutant (MUT) and wild type (WT) mice, MBL-A was mainly seen as tetramers, trimers and dimers, when serum samples were separated under non-reducing and non-heat-denaturing conditions on SDS-PAGE and immunoblotted. Only small amount of monomers (1×3) was present in serum. The migration positions of covalent oligomeric forms of MBL-A are indicated on the right. Band marked with 2 represent two-chain covalent species of polypeptides. Representative samples are shown. (B) The distribution of the MBL-A oligomers was unchanged in serum of 2 months old female LH mutant mice compared with wild type (n = 4 WT, 4 MUT). Intensity of MBL-A oligomers was quantified from immunoblots and the level of oligomeric form was calculated as a proportion of total intensity of MBL-A. The values represent the average ± SD of the serum samples. (C) The elution profile of MBL-A in serum of 1 year old LH mutant mice was quite similar to the elution profile of wild type mice. The oligomeric forms were separated with gel filtration chromatography and quantified from immunoblots. The average elution profiles of three mice (WT and MUT) are shown. The elution positions of molecular weight markers are indicated.</p

Amount of MBL-A in serum and liver of LH mutant mice.

<p>(A) Amount of MBL-A was reduced in the serum of LH mutant mice (MUT) compared with the corresponding wild type mice (WT). Amount of MBL-A was analyzed from the serum of the 2 months old male (n = 4 WT, 4 MUT) and female (n = 4 WT, 4 MUT) and 1 year old male (n = 4 WT, 3 MUT) and female (n = 5 WT, 6 MUT) by immunoblots. The values represent the average ± SD of the samples. MBL-A monomers in the serum of 2 months old female (B) and 1 year old male (C) wild type and LH mutant mice did not show significant differences in electrophoretic mobility. (D) The immunoblot analysis of liver homogenate (40 µg of soluble protein) of wild type and LH mutant male mice did not show differences in mobility of MBL-A monomers. (E) The amount of MBL-A in liver of LH mutant mice is higher than in wild type. Amount of MBL-A in liver was analyzed with immunoblots and α-tubulin was used as control to normalize the quantities of protein. In immunoblots B, C and D two representative wild type and LH samples are shown. MBL-A in LH mutant mice does not show doublet band, differing from the results of rat MBL-A overexpressed in cell culture system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113498#pone-0113498-g001" target="_blank">Fig. 1A</a>). The values in figure A and E represent the average ± SD of the samples. P values were calculated using unpaired homoscedastic student t-test with two-tailed distribution. * p<0.05.</p

Mass spectrometry identification of peptides and modifications from tryptic digests of recombinant rat MBL-A.

<p>Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = full length LH3; LH3-N = amino-terminal fragment of LH3; MUT = LH mutant; Hyl = hydroxylysine; Gal = galactosyl; Glc = glucosyl; Hyp = hydroxyproline.</p><p>Glc-Gal-Hyl 340 Da, Gal-Hyl 178 Da, Hyl/Hyp 16 Da.</p><p>Mass spectrometry identification of peptides and modifications from tryptic digests of recombinant rat MBL-A.</p

Oligomer distribution of recombinant MBL-A is altered in LH3^−/− knockout MEF cells.

<p>(A) Recombinant rat MBL-A produced in LH3 manipulated MEFs was separated under non-reducing and non-heat-denaturing conditions on SDS-PAGE and immunoblotted. Different oligomeric pattern was seen in MBL-A produced in LH3^−/− knockout MEFs compared with the other samples. The migration positions of covalent oligomeric forms of MBL-A are indicated on the right. Band marked with # represents four-chain covalent species of polypeptides. Representative samples are shown. (B) Quantification of MBL-A bands from immunoblots confirmed the change in the oligomer distribution of MBL-A produced in LH3^−/− knockout MEFs. Intensity of MBL-A band around molecular marker 150 kDa (marked with #) was quantified instead of the 2×3 band in LH3^−/− knockout MEFs. The values represent the average ± SD of six to eight experiments. (C) The oligomeric forms of recombinant rat MBL-A produced in LH3 manipulated MEFs were separated with a gel filtration chromatography and quantified from immunoblots. The gel filtration elution profile of MBL-A produced in LH3^−/− knockout MEFs also differed from wild type. Double transfection with MBL-A and LH3 constructs normalized the elution profile in LH3^−/− knockout MEFs. Equal volumes of concentrated cell culture media were used in all analysis. The average elution profiles of four to five experiments are shown. The elution positions of molecular weight markers are indicated. Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = full length LH3; LH3-N = amino-terminal fragment of LH3; MUT = LH mutant.</p

Sputum Proteomics Identifies Elevated PIGR levels in Smokers and Mild-to-Moderate COPD

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood

Sputum Proteomics Identifies Elevated PIGR levels in Smokers and Mild-to-Moderate COPD

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood

The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin

<div><p>Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.</p> </div

The distribution of adiponectin oligomers is altered in the serum of LH mutant mice.

<p>(A) The ratio of HMW/total adiponectin was significantly increased and MMW/total decreased in serum of 2 months old male mice, even though the total amount of adiponectin was lowered as seen in the elution profile of adiponectin (B). The oligomeric forms were separated with gel filtration chromatography and quantified either from adiponectin immunoblots (A) or by ELISA (B). The level of oligomeric forms (A) was calculated as a proportion of total adiponectin. The values represent the average ± SD of the experiments. P values were calculated using unpaired homoscedastic student t-tests with two-tailed distribution.* p<0.05, ** p<0.01, *** p<0.001.</p

The expression of genes involved in β-oxidation, fatty acid synthesis and gluconeogenesis in LH mutant mice.

<p>Quantitative RT-PCR was used to determine changes in the gene expressions in LH mutant mice after 4 months of high fat diet. The columns represent the relative expression levels of muscular genes involved in the mitochondrial β-oxidation; CPT-1 and VLCAD and hepatic genes involved in the mitochondrial β-oxidation, fatty acid synthesis and gluconeogenesis; VLCAD, ACACA and PEPCK-C, respectively. For relative quantification of gene expression, the results were normalized using GADPH and β-actin as endogenous controls, and the expression of the wild type samples were set to 1. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050045#s3" target="_blank">Results</a> represent means with 95% confidence interval of 10 independently analyzed mice for each genotype. P values were calculated using unpaired heteroscedastic student t-test with one-tailed distribution. * p<0.05, ** p<0.01, *** p<0.001. Abbreviations: CPT-1 = carnitine palmitoyltransferase, VLCAD = very long chain acyl-CoA dehydrogenase, Acaca = acetyl-CoA carboxylase, PEPCK-C = phosphoenoylpyruvate carboxykinase.</p

Distribution of recombinant adiponectin in MEF cell lysate and cell culture media.

A<p>Average of three experiments in which equal number of cells were used.</p>B<p>The results are calculated as a percentage of adiponectin from the total adiponectin on a cell culture plate (ng in lysate+ng in media measured by ELISA) and presented as average values ± S.D. from 3 different transfections.</p