Question

Describe the steps in the synthesis and transport of a soluble, N-glycosylated glycoprotein from when is emerges from the ribosome during translation, until it is secreted at the cell surface. Be sure to include glycan synthesis and transfer to the protein.

I have seen this question been posted earlier, but I was wondering if someone could "dumb" it down for me as I find the process quite confusing.
Thank you in advance!

Answer 1

Steps in the synthesis and transport of a soluble, N-glycosylated glycoprotein from when is emerges from the ribosome during translation, until it is secreted at the cell surface:-

There are many types and mechanisms of glycosylation, and the most common type is N-glycosylation. Glycosylation is often characterized as a post-translational modification. While this is true with other types of glycosylation, N-glycosylation often occurs co-translationally, in that the glycan is attached to the nascent protein as it is being translated and transported into the ER. The "N" in the name of this type of glycosylation denotes that the glycans are covalently bound to the carboxamido nitrogen on asparagine (Asn or N) residues.

Because the ER is the site of translation and processing of most membrane-bound and secreted proteins, it is not surprising that most of these are N-linked glycoproteins. Besides being the most common type of glycosylation (90% of glycoproteins are N-glycosylated), N-linked glycoproteins also have large and often extensively branched glycans that undergo multiple processing steps after being bound to proteins.

N-glycosylation is conserved across eukaryotes and archae, and a considerable number of the enzymes and processes involved are also conserved across the different species .N-glycosylation can be broken down into separate events, as follows:

• Precursor glycan assembly
• Attachment
• Trimming
• Maturation

As described previously, different enzymes are required for each step in during glycosylation, which facilitate diversity in the glycans that are generated , But N-glycosylation initially occurs identically for all proteins, and the diversity does not manifest until the subsequent trimming and glycan maturation.

Precursor glycan assembly :-

Oligosaccharides attached via N-glycosidic linkages are derived from a 14-sugar precursor molecule comprised of N-acetylglucosamine (GlcNAc), mannose (Man) and glucose (Glc). These sugars are added consecutively onto dolichol, a polyisoprenoid lipid carrier embedded in the ER membrane . The first 7 sugars are donated from sugar nucleotides (UDP- and GDP-sugars) in the cytoplasm and bound to dolichol via a pyrophosphate linkage (-PP-). After the Man5GlcNAc2-PP-dolichol intermediate is completed, the entire complex is flipped into the lumen of the ER, after which the final 7 sugars are donated from Man- and Glc-P-dolichol molecules to make the Gcl3Man9GlcNAc2-PP-dolichol precursor glycan.

Glycan attachment :-

Glycosylation is often characterized as a post-translational modification. While this is true with other types of glycosylation, N-glycosylation often occurs co-translationally, in that the glycan is attached to the nascent protein as it is being translated and transported into the ER. The "N" in the name of this type of glycosylation denotes that the glycans are covalently bound to the carboxamido nitrogen on asparagine (Asn or N) residues.

Because the ER is the site of translation and processing of most membrane-bound and secreted proteins, it is not surprising that most of these are N-linked glycoproteins. Besides being the most common type of glycosylation (90% of glycoproteins are N-glycosylated), N-linked glycoproteins also have large and often extensively branched glycans that undergo multiple processing steps after being bound to proteins.

One aspect to note is that not all Asn residues with the predicted consensus sequence are glycosylated. N- to C-terminal protein synthesis results in transport of the growing polypeptide into the ER in the same orientation, and protein folding occurs soon after the polypeptide enters the ER. Therefore, as protein folding increases, OSTase is less able to access the consensus sequence for glycan transfer. Indeed, more N-terminal Asn residues are glycosylated than C-terminal Asn residues.

Glycan trimming in the ER :-

Oligosaccharides are trimmed in both the ER and Golgi by glycosidases via hydrolysis. Glycan trimming in the ER, though, serves a different purpose than trimming in the Golgi.

In the ER, sugar hydrolysis is used to both monitor protein folding and indicate when proteins should be degraded. Glucosidases I and II remove 2 terminal Glc from the precursor glycan, after which calnexin and calreticulin, which are membrane-bound and soluble (respectively) sugar-binding lectins, bind to the nascent glycoprotein via the remaining Glc and act as chaperones to help the protein fold properly. The final Glc is soon hydrolyzed by glucosidase II, releasing the glycoprotein from the chaperone. Non-native–folded proteins are recognized by UDP-glucose glycoprotein glucosyltransferase, which transfers a Glc to the glycoprotein, and the protein again is bound to the lectin chaperones to facilitate proper protein folding . This cycle of Glc addition and removal continues until the protein is correctly folded, at which time it is not reglycosylated, and the glycoprotein is trafficked to the Golgi for further processing . The glycan structure for all properly folded glycoproteins that proceed to the Golgi is Man9GlcNAc2 in higher eukaryotes.

An ER-resident mannosidase (ERManI) plays a key role in identifying proteins that are unable to fold properly. Proteins that lose 3–4 mannose residues in the ER via ERManI activity are transported out of the ER and deglycosylated by glycanase N (removes the entire glycan en bloc) and delivered to ER-associated degradation (ERAD) . It is thought that ERManI acts as a timer of sorts, because it has a slow rate of mannose hydrolysis that allows nascent proteins multiple rounds of reglycosylation to attempt to fold properly before mannose residues are removed and the protein is targeted for degradation .

Glycan maturation in the Golgi :-

To this point during glycosylation, all N-linked glycoproteins have the same precursor glycan structure. Glycan processing in the Golgi apparatus combines both trimming and adding sugars to diversify the glycans on individual glycoproteins. As with precursor glycan biosynthesis, this maturation pathway to generate diverse oligosaccharides is highly ordered, such that each step is dependent upon the previous step. To this end, the Golgi segregates specific enzymes into different cisternae to facilitate this step-wise process.

The final glycan structures can be broadly separated into two groups:

• Complex oligosaccharides—contain multiple sugar types
• High-mannose oligosaccharides—multiple mannose residues
• Hybrid—branches of both high mannose and complex oligosaccharides

Glycans destined to be complex oligosaccharides are trimmed by Golgi mannosidase I and II and glycosylated by GlcNAc transferase, resulting in a common core region . The core then becomes the substrate for multiple Gtfs that consecutively transfer sugar moieties from sugar nucleotides to build variable-length and -branched oligosaccharide chains of GlcNAc, galactose (Gal), N-acetylneuraminic acid (NANA or sialic acid) and fucose. Any glycoproteins that progress through this processing from the common core stage become resistant to glycan removal by endoglycosidase H (endo H), which is used experimentally to determine if glycoproteins contain high-mannose or complex oligosaccharides.

Unlike complex oligosaccharides, high-mannose oligosaccharides do not carry other sugar moieties, although some of the Man residues are often trimmed by Golgi mannosidase I. Whether a glycan is processed into a complex oligosaccharide rather than remaining a high-mannose oligosaccharide is dependent upon the accessibility of the processing enzymes to the glycan, which can be hindered by the glycoprotein conformation. Some glycoproteins have hybrid oligosaccharides, comprising a combination of complex and high-mannose glycans.

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