Question

Briefly discuss why multiple related N-glycan isomers (often displaying exactly the same monosaccharide compositions but in slightly different configurations) are commonly found to be linked to glycoproteins such as bovine and human lactoferrin.

Answer 1

The amino acid composition of human and bovine lactoferrin has a 78% homology. Despite the similarity in human and bovine LF, human LF has three potential glycosylation sites (Asn138, Asn479, Asn624) while bovine LF has 5 N-glycosylation sites (Asn233, 281, 368, 476, and 545). All these N-glycans are attached to Asparagine amino acid via N-acetylglucosamines (HexNAc). Of these sites, hLF has two common glycosylation sites- Asn 138 and Asn 479 while bLF has 4 common glycosylation sites-Asn233, 368, 476, and 545. The core of N-glycan has two GlcNAc and three mannose residues linked by N-glycosidic bonds. Other enzymes will extend this core with monosaccharides, which determines the degree of branching as well as the linkages present. NeuAC and Fuc can be used for elongation which increasing the complexity of the structure.

13 N-glycans are common in humans and bovine species. Human LF has 18 unique N-glycans while bovine species have 16 unique N-glycans. The basic unit however will be a pentasaccharide core. NeuGc is only present in bovine LF but absent in human LF. Human LF has higher fucosylation than bovine LF while the sialyation is lesser than bovine LF. Bovine LF has more mannose present in N-glycans. These different glycan patterns are thought to be important for immunogenicity and allergic reactions. When human LF is expressed in transgenic cows (bovine species), it expresses a different glycosylation pattern than what it would express in humans. N-Glycosylation occurs mostly in ER of the cell. Hence, these differences in N-glycosylation are due to post translational modification and are organism specific.

These N-glycans may be involved in host defense against pathogens. They may be involved in recognition of lectins on the cell surface of the pathogen. Human LF plays an important role in protection of intestinal mucosa from pathogens in breast fed babies. Hence, the glycosylation pattern undergoes variation as lactation progresses in humans. The protein bound glycans are thought to act as decoy for pathogens. The glycosylation of bLF at Asn281 protects it from degradation from trypsin. N-glycosylation is not involved in protection from trypsin of human LF, despite it being more resistant to trypsin than bLF. The main role of N-glycans and their differences in human and bovine LF seems to be in protection against bacterial and viral infections. If Fuc is removed from human LF in vitro, then it cannot bind to small intestine in monkeys. N-glycans are also part of antigenic determinant of LF and thus, affect immunogenicity.