Researchers in Japan believe they have found a way to make glycosylation predicable and controllable. A molecular code embedded in the lysosomal-associated membrane protein 1 (LAMP-1) that induces the Lewis X glycan structure is the key.

“This molecular code consists of a sequence of 29 contiguous amino acid residues, which could control the glycosylation of erythropoietin (a typical biopharmaceutical glycoprotein) simply by connecting it to the end of the molecule,” says corresponding author Koichi Kato, PhD, at the National Institutes of Natural Sciences (NINS) in Okazaki. This 29-amino acid sequence also can be embedded into other glycoproteins or removed by enzymatic treatment, paving the way for more deliberate control of glycosylation in mammalian cells used in the production of biopharmaceuticals.

Glycosylation is a key step in biomanufacturing

Glycosylation is widely used in biopharmaceutical development and “has a decisive influence on their efficacy and safety as drugs,” Kato says. “However, as information on glycan structures of proteins is not directly encoded in the genome, it has been considered difficult to control their glycosylation.” Unpredictable glycosylation profiles, therefore, are common. This “Lewis X code” may remove that unpredictability.

More specifically, writing in Communications Biology, Kato and colleagues at Nagoya City University showed that a contiguous sequence of 29 amino acids in the N-terminal domain of LAMP-1 (from Ile136 to Asn164) with no N-glycosylation sites promotes Lewis X modification through fucosyltransferase 9 (FUT9). By attaching that amino acid sequence to the C-terminus of the erythropoietin they used as a model glycoprotein, they achieved Lewis X modification.

As their research demonstrated, “Other cellular components were dispensable for inducing the observed modification,” they wrote. This suggests that the amino acid sequence from LAMP-1 is a key molecular code that can be used with other glycoproteins for Lewis X modification including, as they demonstrated, into fetuin (a protein that carries fatty acids in the bloodstream).

None-the-less, Kato is cautious. “It remains to be investigated to what extent this glycosylation control code can be universally applied to other glycoproteins,” he notes.

The paper also noted the possibility of other, as yet unidentified, codes for specific N-glycosylation in proteins. “In the future,” Kato points out, “it is possible that novel molecular codes will be discovered one after another by studying various glycoproteins. By using them as control codes for glycosylation, we hope to contribute to the development of biopharmaceuticals and the advancement of cellular medicine.”

To learn more about glycosylation approaches and strategies, see GEN: “Optimize Protein Glycosylation–and Yield–by In Vitro Glycoengineering,” “Building Therapeutic Proteins through Plant Glycosylation: A Sweet Solution,” Applying the Bioprocessing 4.0 Paradigm to Antibody Glycosylation,” and “Getting at Glycosylation.”