N-glycomic Analysis of Z(IgA1) Partitioned Serum and Salivary Immunoglobulin A by Capillary Electrophoresis

Abstract

Aims: To apply capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions. Background: Altered N-glycosylation of body fluid glycoproteins can be effective indicators of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant of the immunoglobulins and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces such as in saliva. Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders. Materials: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions. Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand six structures were found in the serum samples not present in the saliva. Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use.

N-Glycosylation Alteration of Serum and Salivary Immunoglobulin A Is a Possible Biomarker in Oral Mucositis

“…Chemicals and reagents, Z(IgA1) antibody production, expression and purification, serum and salivary IgA capturing, N-glycan release and fluorophore labeling, exoglycosidase based carbohydrate sequencing and capillary electrophoresis analysis were applied as described in detail in our recent paper [ 14 ].…”

“… Fourteen serum ( a ) and six salivary ( b ) IgA glycoforms showed significant changes during autologous peripheral stem cell transplantation (APSCT). The abbreviations of the Oxford nomenclature were applied as referred to in our previous work [ 14 ]. The plots show the distribution pattern at various APSCT time points in patients and in controls (* p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001).…”

Female sex as an independent prognostic factor in the development of oral mucositis during autologous peripheral stem cell transplantation

“…Sample collection was based on the protocols described previously 14 , 15 . In brief, following oral cavity rinse with 25 ml of physiological saline solution (B. Braun Melsungen AG, Melsungen, Germany) for 30 s, saliva was collected for 5 min in 15 ml disposable, pre-disinfected tubes (Sigma-Aldrich, St. Louis, MO, USA).…”

Salivary Osteopontin as a Potential Biomarker for Oral Mucositis

“…Blood and Unstimulated Whole Saliva (UWS) samplings were performed at the same time point (between 7 a.m. and 8 a.m., taking into consideration the diurnal rhythm of saliva constituents) on specified days of the peritransplantation period as follows: hospital admission and start of the conditional therapy (day −3/−7), transplantation day, before administering the harvested CD34+ stem cells in a stem cell infusion (day 0), the day of usually the deepest point of cytopenia with most severe oral mucositis (day +7), the day of neutrophil and thrombocyte engraftment if insertion and proliferation of stem cells were successful (day +14), based on the protocols described in details previously [ 2 , 3 , 4 ]. Briefly, blood samples were collected into BD Vacutainer Blood Collection Tubes using a clot activator (BD, Franklin Lakes, NJ, USA), centrifuged at 1200× g for 30 min and the serum fractions were stored at −70 °C until further processing within one hour of collection.…”

“…Saliva is one of the most important pillars of oral immunity and a special mirror of the body’s defence mechanisms [ 1 , 2 , 3 , 4 ]. As a rich reservoir of proteins, especially glycoproteins, electrolytes, lipids and other molecules with several biological functions, saliva plays a pivotal role in the maintenance of oral homeostasis and mucosal integrity [ 5 ].…”