BIOTECHNOLOGY
Te predicted molecular weight of
a protein can be easily determined, for example using one of a range of tools available, including free online tools such as ExPASy, to calculate the sum of the molecular weights of all amino acids comprising that protein. However, the calculated molecular weight is invariably different from that observed on the Western blot. Here, we summarise the most common reasons why this may occur.
POST-TRANSLATIONAL MODIFICATIONS Glycosylation and glycanation. Most proteins that are synthesised on ribosomes associated with the endoplasmic reticulum undergo glycosylation, where sugar moieties are covalently attached to the polypeptide chain. Te two most common types of glycosylation in eukaryotes are N-linked glycosylation (to asparagine), and O-linked glycosylation (to serine and threonine). Extensive glycosylation increases molecular weight, slowing protein migration on a Western blot, but is not accounted for in a molecular weight calculation based on protein sequence (Figs 1 and 2). Enzymatic de-glycosylation is an experimental technique commonly used to verify whether a studied protein is glycosylated. Prior to Western blotting, the protein sample is incubated with an enzyme that is able to remove part or full glycan chains. Protein species from the digested sample are then compared with the undigested sample, and any observed shift in molecular weight indicates protein glycosylation. One commonly used enzyme is PNGase F, which removes N-linked glycans by cleaving the bond between the innermost N-Acetylglucosamine of the glycan chain and the asparagine residue. Proteoglycans are a special case group
of glycoproteins. Tese extracellular matrix proteins have long, unbranched glycosaminoglycan chains, covalently attached to the amino peptide chain core. Usually, the molecular weight of the sugar group is even larger than the protein component. Fig. 1 shows programmed cell death ligand 1 (PD-L1, CD274, or B7-H1) (66248-1-Ig), a type I transmembrane protein, acting as a key regulator of the adaptive immune response. Full-length PD-L1 molecular
Fig. 1
weight is 33 kDa. Te signal peptide is cleaved off during protein transport to the plasma membrane, and the protein is heavily N-glycosylated with an apparent molecular weight of 45–70 kDa, with the major glycosylated form of 45–50 kDa (PMID: 27572267). CD133, also known as PROM1
(prominin-1) (18470-1-AP), is a transmembrane glycoprotein with an NH2-terminal extracellular domain, five transmembrane loops and a cytoplasmic tail. Te protein is highly glycosylated with an apparent molecular weight of 115–120 kDa. After treatment with PNGase F, CD133 shifts to a protein with a molecular weight of 75–85 kDa, which corresponds to the calculated molecular weight of de- glycosylated CD133 (PMID: 23150174). Fig. 2 shows Decorin (14667-1-AP), which is a member of the small leucine- rich proteoglycan family of proteins, the precursor of which forms a range of
43–47 kDa molecular weight proteins. It contains a cleavable N-terminal peptide signal and can also be glycosylated. Te attachment of glycosaminoglycans (chondroitin sulphate or dermatan sulphate) to decorin occurs in the Golgi apparatus prior to secretion of the mature glycanted form from cells. Phosphorylation. One of the most common post-translational modifications is phosphorylation. Taking place on serine, threonine and tyrosine residues, phosphorylation is catalysed by phosphatases, regulating protein function, enzymatic activity, protein-protein interactions and protein localisation. Although the addition of a single phosphoryl group adds just +/-1 kDa to the molecular weight, which is often beyond the resolution of standard SDS- PAGE, phosphorylation at multiple sites can lead to more noticeable molecular weight changes (Fig. 3).
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