Sequence characterization and N-glycoproteomics of secretory immunoglobulin A from donkey milk B.S. Gnanesh Kumar a, ⁎, Apoorva Rawal b a Department of Biochemistry, CSIR-Central Food Technological Research Institute (CFTRI), Mysuru 570020, Karnataka, India b Devi Ahilya Vishwavidyalaya, Takshashila Campus, Indore 452017, Madhya Pradesh, India abstract article info Article history: Received 17 December 2019 Received in revised form 31 March 2020 Accepted 31 March 2020 Available online 3 April 2020 Keywords: Donkey milk Immunoglobulin A N-glycosylation Secretory immunoglobulin A (sIgA) is the major antibody present in the human milk where it confers passive im- munity to neonates. Other than human, non-ruminants such as equine, swine etc., also possess sIgA in milk but detailed characterization is limited. In the present study, we characterized sIgA from donkey milk for amino acid sequence and N-glycosylation through LC-MS/MS analysis. The complete amino acid sequence of alpha chain constant region (C H ) was elucidated. The sequence analysis of variable regions (V H and V L ) and light chain con- stant region (C L ) showed several amino acid substitutions indicating the presence of diverse immunoglobulin repertoire. Glycoproteomic analysis of secretory component revealed bi-antennary complex and hybrid types with differential core fucosylation at site N 83 LT, only complex glycans at N 135 GT, N 423 GT and N 530 LT with mainly NeuAc whereas N 291 QT harbors high mannose glycans. Heavy chain possesses majorly bi-antennary complex with differential core fucosylation at sites N 139 AS and N 338 VS, in which N 338 VS shows partial occupancy. Joining chain harbors only complex type at N 72 IS, with core fucosylation and terminal NeuGc to some extent. N-glycan repertoire in part is similar to human sIgA. This comprehensive analysis of sequence and glycan pattern of donkey milk sIgA would be beneficial for its potential applications. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Secretory IgA (sIgA) is the major antibody in the mucosal secretions of most mammals [1]. It is also predominantly present in colostrum and milk of human, horse, swine, etc., where the concentrations vary ac- cording to lactation period [2–5]. The presence of sIgA in milk provides passive immunity to neonates by neutralizing the broad spectrum of pathogens and toxins in the gastrointestinal tract [6]. Human sIgA is well characterized and is a dimer composed of 4 heavy chains (HC, ~55 kDa) and 4 light chains (LC, ~25 kDa) along with a joining chain (JC, ~16 kDa) that connects dimers and a secretory component (SC, ~75 kDa) which helps in transport of IgA across epithelial cells [7]. In general, heavy and light chain contains two distinct domains; the N- terminus variable (V H and V L ) and C-terminus constant (C H and C L ) re- gion. The variable region is responsible for antigen binding and wide array of antigen-specific variable regions are generated through somatic recombination with diversity (D) and joining (J) segment i.e. V(D)J in heavy chain and only V(J) in light chain. Additionally, the hyper muta- tion and gene conversion increases the variable region diversity [8]. In humans, two subtypes of alpha constant region are known i.e. IgA1 and IgA2, whereas horse possess single alpha constant region. However, multiple light chain constant genes are observed in many mammals in- cluding human and horse [9–11]. N-glycosylation is the major posttranslational modification of milk proteins including immunoglobulins, wherein oligosaccharides are co- valently linked to asparagine in a sequence motif Asn-X-Thr/Ser/Cys [12]. Human sIgA is heavily glycosylated with N-glycans in SC, HC and JC. Depending on the IgA subtypes the number of glycosylation sites in heavy chain differs and also O-glycosylation has been observed in IgA 1 [13]. Glycans exhibit structural variants at each site and are essential for effector functions of immunoglobulins [14]. The glycans on sIgA are involved in specific interactions with microbes that prevent adhe- sions in gastrointestinal tract resulting in the reduced infections and may also function as prebiotics to promote growth of beneficial bacteria in the gut [15,16]. N-glycosylation analysis of human milk sIgA has re- vealed the large structural heterogeneity at each site. N-glycans ranging from bi, tri/tetra antennary complex type with differential core fucosylation and sialylation with N-acetyl neuraminic acid (NeuAc) are observed [17,18]. Despite sIgA is the major antibody in the milk of equine there are no reports on its characterization. Donkey milk is the best substitute for human milk, wherein the ther- apeutic properties are well documented since ancient times [19,20]. Several studies have focussed on understanding the nutritional compo- sition and potential health benefits of donkey milk that has revealed the similarity in lactose and protein content compared to human milk. International Journal of Biological Macromolecules 155 (2020) xxx ⁎ Corresponding author. E-mail address: gnanesh@cftri.res.in (B.S. Gnanesh Kumar). BIOMAC-15204; No of Pages 9 https://doi.org/10.1016/j.ijbiomac.2020.03.253 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac Please cite this article as: B.S. Gnanesh Kumar and A. Rawal, Sequence characterization and N-glycoproteomics of secretory immunoglobulin A from donkey milk, , https://doi.org/10.1016/j.ijbiomac.2020.03.253