Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions.

Background

The carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins.

Results

The results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.

Conclusions

Together, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.