Investigation of the glycosylation profile of IgG and IgA in saliva and plasma of healthy individuals using liquid chromatography coupled to mass spectrometry (LC-MS)

Glycosylation is an enzymatic process that attaches glycans, i.e. sugars, to
proteins, lipids and other biomolecules. Glycosylation is one of the most
common posttranslational modifications of proteins. It can influence protein
folding, stability, localization, and activity. One of the most common
glycosylation types in humans is N-glycosylation, where glycans are attached to
the nitrogen atom of an asparagine residue within the carrier protein. This
takes place in the endoplasmic reticulum and the glycan chain is further
processed in the Golgi apparatus [1]. This research project will focus on
N-glycosylation of immunoglobulins.

Immunoglobulins (Ig*s) are a family of glycoproteins that are produced by
B-cells upon detecting antigens. They can be divided in two heavy chains and
two light chains. Differences in the heavy chain of the glycoproteins define
five classes of Ig*s that have a different function in the immune system [2].
From the five classes of Ig*s there are several that are present in saliva,
i.e. IgG, IgM, IgE and IgA [3-4]. This project will focus on IgG and IgA, since
IgM and IgE concentrations are very low in saliva [3-4].
Immunoglobulin G (IgG) can be divided in four subclasses (IgG1, IgG2, IgG3 and
IgG4) and is found in internal body fluids such as blood and lymph. In its
fragment crystallizable (Fc) region, IgG has N-linked glycosylation at
asparagine-297, which is important for the interaction with Fc gamma receptors
[5]. Immunoglobulin A (IgA) has two different subclasses, IgA1 and IgA2, and
can occur as a dimer. IgA is mainly found in secretions such as saliva, tears,
milk, sweat and in the lumen of the gut, but small amounts are also present in
blood. Two N-glycans are present on IgA1, while IgA2 carries four [6]. The main
function of IgA and IgG is the neutralization and opsonization of pathogens and
complement activation [2].

1. Shylaja M and Seshadri H.S. (2010).Glycoproteins: an overview. Biochemical
Education 17(4) 17:170-78
2. Parham, P. (2009). The Immune system (3rd ed.). London and New York, United
Kingdom and United States of America: Garland Science.
3. Sandra Pineda-Martínez, José Luis Hernández-Islas, Mónica Patricia
Escobedo-Torres, Iris Evelin Paredes-Alonzo, Carlos López-Candiani, Dolores
Correa, Marcela Vela-Amieva. (2015). Immunoglobulin Concentrations in Plasma
and Saliva During the Neonatal Period. Pediatrics & Neonatology
S1875-9572(15)00145-X
4. Mario R. Romero, Marta L Lozano, Carolina Posada, Paola A Rueda, Nelly S.
Roa, Adriana Rodríguez. (2011). Immunoglobulin A, G and M levels in Saliva in
Children Between 3-12 Years of Age, Healthy and with Gingivitis. Acta Odontol.
Latinoam. 24(2): 176-182
5. Bondt A, Selman MH, Deelder AM, Hazes JM, Willemsen SP, Wuhrer M, Dolhain
RJ. (2013). Association between galactosylation of immunoglobulin G and
improvement of rheumatoid arthritis during pregnancy is independent of
sialylation. Journal of Proteome Research;12(10):4522-31
6. Jerrard M. Hayes, Eoin F. J. Cosgrave, Weston B. Struwe, Mark Wormald, Gavin
P. Davey, Roy Jefferis and Pauline M. Rudd. (2014). Glycosylation and Fc
Receptors. Current Topics in Microbiology Immunology; 382:165-99

We want to study the glycosylation profiles of IgG and IgA in plasma and
saliva of healthy volunteers, collected at the same moment. By investigating
the differences between glycosylation profiles of both salivary and plasma IgG
and IgA, we want to assess the suitability of saliva collection for the study
of glycosylation of these two proteins in biomarker research.

To investigate the glycosylation of saliva and plasma in healthy individuals,
blood and saliva samples will be collected from 30 healthy individuals aged
18-50 years. Purification of IgG and IgA will be accomplished by using
immobilized affinity ligands that are coupled to magnetic beads. The affinity
ligands specifically bind to and isolate IgA or IgG from other compounds
present in saliva and plasma. A similar method using protein G sepharose beads
is already established for IgG in plasma, but needs to be adjusted for IgG in
saliva and IgA in saliva and plasma. Purified IgA and IgG will then be
digested by a proteolytic enzyme (trypsin) enabling the separation of the
different (glyco)peptides by liquid chromatography and the profiling of their
glycosylation by mass spectrometry. Specialized, in-house built software
(LaCyTools) will be used for LC-MS data analysis and the visualization of
results.

Collection of blood is approximately around the 5 ml. This will not have any
adverse consequences for the participant as the amount of blood drawn is
approximately 1/100 of what is normally collected during blood donations.
Collection of saliva will not have any adverse consequences for the participant
as the collection of saliva is not invasive and easy to collect.