| Genetic manipulation at the DNA level has revolutionized modern biotechnology. The recent completion of the human genome sequence will significantly facilitate the identification of genes and gene products that are involved in various physiological or pathological states. The demand for protein-based therapeutic products is expected to increase in the near future, many of them being glycoproteins. Choosing the right host for the production of recombinant human glycoproteins could significantly affect the cost of production. Insect cell expression systems with the ability to perform post-translational modifications, to attain higher yields at lower cost offer very promising alternatives to the mammalian cell cultures. Unfortunately, the N-glycan expressed in insect cells generally are either of high mannose or truncated, low mannose type, mono- or bi-fucosylated on the innermost GlcNAc. Most importantly, they are mostly not sialylated.; In this work, the capacity of insect cells for protein glycosylation was evaluated. A key enzyme in the elongation of N-glycans, β- N-acetylglucosaminyltransferase I (GnT-I), is abundant but the next elongation enzyme, β-1,4-galactosyltransferase (β4GalT-I), was not detected in Spodoptera frugiperda and was at a very low level in Trichoplusia ni cell line. However, the occurrence of GlcNAc terminated structure is rare in recombinant N-glycans. It was speculated that insect cells contain a membrane-bound β- N-acetylglucosaminidase, which removes GlcNAc that was attached on Manα(1-3)-arm to prevent elongation. Most interestingly, there is abundance of β-N-acetylglucosaminidase secreted into the culture media in both Sf9 and High Five cell cultures. β- N-Acetylglucosaminidases from both cells were purified, and evaluated for their specificity towards GlcNAc-terminated N-glycans and chitooligosaccharides. Sf9 β-N-acetylglucosaminidase secreted into the media was able to digest both N-Glycan as well as chitooligosaccharides. However, the secreted β-N-acetylglucosaminidase from Trichoplusia ni was essentially an exo-type chitinase, digesting GlcNAc from the non-reducing terminal. We propose that the Sf9-secreted β- N-acetylglucosaminidase is directly involved in accumulation of low mannose (truncated) N-glycans in recombinant glycoprotein.; In addition, a genomic approach was used to identify Drosophila- N-acetylglucosaminidase from the Drosophila genome databank using a Bombyx mori and Manduca sexta β- N-acetylglucosaminidase as a query sequences. Two putative genes, which share 33% and 35% sequence identity with human lysosomal β- N-acetylglucosaminidase respectively were identified, cloned, expressed and evaluated for their natural substrate. Both enzymes which share 36% identity to each other are chitinases. |
