Protein and site specificity of fucosylation in liver-secreted glycoproteins.

Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.

Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of (18)O water. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease.

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2-3,6,8)-neuraminidase and ß(1-4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior.

Site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma.

Haptoglobin is a liver-secreted glycoprotein with four N-glycosylation sites. Its glycosylation was reported to change in several cancer diseases, which prompted us to examine site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. To this end, we have used two-dimensional separation composed of hydrophilic interaction and nano-reverse phase chromatography coupled to QTOF mass spectrometry of the enriched glycopeptides. Our results show increased fucosylation of haptoglobin in liver disease with up to six fucoses associated with specific glycoforms of one glycopeptide. Structural analysis using exoglycosidase treatment and MALDI-MS/MS of detached permethylated glycans led to the identification of Lewis Y-type structures observed particularly in the pooled hepatocellular carcinoma sample. To confirm the increase of the Lewis Y structures observed by LC-MS, we have used immunoaffinity detection with Lewis Y-specific antibodies. The presence of multiply fucosylated Lewis Y glycoforms of haptoglobin in the disease context could have important functional implications.

An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization.

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens.

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.