Glycosylation is a critical function of the biosynthetic-secretory pathway in the endoplasmic reticulum

(ER) and Golgi apparatus. Glycosylation is the reaction in which a carbohydrateis attached to a
hydroxyl or other functional group of another molecule. In biology, glycosylation is an enzymatic
process that attaches glycans to proteins, lipids, or other organic molecules. This enzymatic process
produces one of the fundamental biopolymers found in cells.

mechanism

Glycosylation is an enzymatic process and it is the most complex posttranslational modification, because of the large number of enzymatic steps
involved. The donor molecule is often an activated nucleotide sugar. The process
is non-templated and the cell relies on segregating enzymes into different
cellular compartments for example endoplasmic reticulum, cisternae in Golgi
apparatus. Therefore, glycosylation is a site-specific modification.

Protein Glycosylation is the post-translational process by which saccharides are

selectively added to specific protein residues utilizing two distinct mechanisms in
order to convey more structural stability or function to the native protein
structure. Specifically this process is necessary for proper modification of a
protein such that it may anchor properly into a phospholipid bilayer or is
conveyed a cell signaling function resultant of the enzyme mediated addition of
sugars in a site-directed manner. However due to the lack of enzyme recognition
or consensus sequence knowledge, the specificity of these mechanisms
occurring on peptide sequences is largely unknown. As such there is a significant
amount of work that has been done in order to define prediction models for
glycosylation sites in order to aid in protein modeling as a whole.
All forms of glycosylation are enzymatic, site specific reactions which utilize an activated
nucleotide sugar. Beyond these three base requirements, several specific types of glycosylation
exist.

question 2
types of Glycosylation

N-linked

Glycan bind to the amino group of asparagine in the ER

O-linked

Monosaccharides bind to the hydroxyl group of serine or threonine in the ER,

Golgi, cystosol and nucleus

Glypiation

Glycan core links a phospholipid and a protein

C-linked

Mannose binds to the indole ring of tryptophan

Phosphoglycosylation Glycan binds to serine via phosphodiester bond

Importance
Glycosylation plays an important role in structure, function, absorption, half-life, clearance, and
safety of therapeutic proteins. Glycosylation is a post-translational modification that is of
paramount importance in the production of recombinant pharmaceuticals as most
recombinantly produced therapeutics are N- and/or O-glycosylated. Being a cell-systemdependent process, it also varies with expression systems and growth conditions, which
result in glycan microheterogeneity and macroheterogeneity. Glycans have an effect on drug
stability, serum half-life, and immunogenicity; it is therefore important to analyze and
optimize the glycan decoration of pharmaceuticals.

question 3
Protein Degradation

Two possible ways

Ubiquitin Pathway:
degrade abnormal proteins and short-lived cytosolic proteins.

Located in cytosol.
ATP dependent

Losysomal pathway:
Degrades long-lived membrane proteins and organelles, for example mitochondria.
ATP-Independent.
Located in lysosome.
Cathepsin

question 4
Besides using rules of thumb, or heuristics, for synthesizing bioseparation
processes, it is often advantageous to consider how two unit operations can be
paired to improve process efficiency. The following section lists some examples
of operations that are logical to pair.
Extraction and Precipitation
The bioproduct is extracted with a solvent and then precipitated. To increase the
yield, it is often desirable to concentrate the extract before the precipitation. The
major hurdle to overcome for this pairing is to find a solvent that will work with
both extraction and precipitation.
Precipitation and Hydrophobic Interaction Chromatography
The pairing of precipitation and hydrophobic interaction chromatography is
usually accomplished for protein purification by using ammonium sulfate to
precipitate impurities, leaving the desired bioproduct in the mother liquor. The
ammonium sulfate is added to a concentration just below that needed to
precipitate the bioproduct. After removal of precipitated impurities, the mother
liquor can be applied directly to a hydrophobic interaction chromatography
column, which was equilibrated to the concentration of ammonium sulfate in the
mother liquor prior to the loading. The bioproduct adsorbs to the column under
these conditions. The column is eluted with a reverse gradient of ammonium
sulfate, and the desired bioproduct is recovered in a fraction from the elution.
Filtration and Extraction
When the bioproduct is contained in the filtrate after filtration, it can often be
extracted with an immiscible solvent. For the extraction of small molecules such
as antibiotics with organic solvents, the pH must usually be adjusted to obtain
the bioproduct in either its free base or free acid form so it will partition into the
organic phase. For the aqueous two-phase extraction of proteins, two polymers
or a salt and a polymer must be added. If the additions to the filtrate can be
made in-line, the filtration and extraction steps can be carried out
simultaneously, reducing the processing time.