The Biosynthesis of NLinked Glycoproteins Involves DolicholPPOligosaccharide

Leloir and his colleagues described the occurrence of a dolichol-pyrophosphate-oligosaccharide (Dol-P-P-oligosaccharide), which subsequent research showed to play a key role in the biosynthesis of N-linked glyco-proteins. The oligosaccharide chain of this compound generally has the structure R-GlcNAc2Man9Glc3 (R = Dol-P-P). The sugars of this compound are first assembled on the Dol-P-P backbone, and the oligosaccharide chain is then transferred en bloc to suitable Asn residues of acceptor apoglycoproteins during their synthesis on membrane-bound polyribosomes. All N-glycans have a common pentasaccharide core structure (Figure 47-5).

To form high-mannose chains, only the Glc residues plus certain of the peripheral Man residues are removed. To form an oligosaccharide chain of the complex type, the Glc residues and four of the Man residues are removed by glycosidases in the endoplas-mic reticulum and Golgi. The sugars characteristic of complex chains (GlcNAc, Gal, NeuAc) are added by the action of individual glycosyltransferases located in the Golgi apparatus. The phenomenon whereby the glycan chains of N-linked glycoproteins are first partially degraded and then in some cases rebuilt is referred to as oligosaccharide processing. Hybrid chains are formed by partial processing, forming complex chains on one arm and Man structures on the other arm.

Thus, the initial steps involved in the biosynthesis of the N-linked glycoproteins differ markedly from those involved in the biosynthesis of the O-linked glycopro-teins. The former involves Dol-P-P-oligosaccharide; the latter, as described earlier, does not.

The process of N-glycosylation can be broken down into two stages: (1) assembly of Dol-P-P-oligosaccha-ride and transfer of the oligosaccharide; and (2) processing of the oligosaccharide chain.

A. Assembly & Transfer of Dolichol-P-P-Oligosaccharide_

Polyisoprenol compounds exist in both bacteria and eukaryotic cells. They participate in the synthesis of bacterial polysaccharides and in the biosynthesis of N-

Sialic acid a2,3 or 2,6

±GlcNAc

Sialic acid a2,3 or 2,6

Sialic acid a2,3 or 2,6

Gal

Gal

Gal

pmJ

Jß1,4

ß1,4|

GlcNAc

GlcNAc

GlcNAc

ß1,2jf

|ß1,2

ß1,2^

Man

Man

Man

! a13N<

Man a1,2

±GlcNAc

Asn Complex

Asn Hybrid

Asn High-mannose

Figure 47-4. Structures of the major types of asparagine-linked oligosaccharides. The boxed area encloses the pentasaccharide core common to all N-linked glycoproteins. (Reproduced, with permission, from Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 1985;54:631.)

Asn Complex

Asn Hybrid

Man "a1,6

GlcNAc

Asn High-mannose

Figure 47-4. Structures of the major types of asparagine-linked oligosaccharides. The boxed area encloses the pentasaccharide core common to all N-linked glycoproteins. (Reproduced, with permission, from Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 1985;54:631.)

linked glycoproteins and GPI anchors. The polyiso-prenol used in eukaryotic tissues is dolichol, next to rubber, the longest naturally occurring hydrocarbon made up of a single repeating unit. Dolichol is composed of 17-20 repeating isoprenoid units (Figure 47-6).

Before it participates in the biosynthesis of Dol-P-P-oligosaccharide, dolichol must first be phosphorylated to form dolichol phosphate (Dol-P) in a reaction catalyzed by dolichol kinase and using ATP as the phosphate donor.

Dolichol-P-P-GlcNAc (Dol-P-P-GlcNAc) is the key lipid that acts as an acceptor for other sugars in the assembly of Dol-P-P-oligosaccharide. It is synthesized

Endoglycosidase F

Endoglycosidase H

Figure 47-5. Schematic diagram of the pentasaccharide core common to all N-linked glycoproteins and to which various outer chains of oligosaccharides may be attached. The sites of action of endoglycosidases F and H are also indicated.

in the membranes of the endoplasmic reticulum from Dol-P and UDP-GlcNAc in the following reaction, catalyzed by GlcNAc-P transferase:

Dol-P + UDP-GlcNAc^ Dol-P-P-GlcNAc+UMP

The above reaction—which is the first step in the assembly of Dol-P-P-oligosaccharide—and the other later reactions are summarized in Figure 47-7. The essential features of the subsequent steps in the assembly of Dol-P-P-oligosaccharide are as follows:

(1) A second GlcNAc residue is added to the first, again using UDP-GlcNAc as the donor.

(2) Five Man residues are added, using GDP-man-nose as the donor.

(3) Four additional Man residues are next added, using Dol-P-Man as the donor. Dol-P-Man is formed by the following reaction:

(4) Finally, the three peripheral glucose residues are donated by Dol-P-Glc, which is formed in a reaction analogous to that just presented except that Dol-P and UDP-Glc are the substrates.

Man a1,3

Figure 47-6. The structure of dolichol. The phosphate in dolichol phosphate is attached to the primary alcohol group at the left-hand end of the molecule. The group within the brackets is an isoprene unit (n = 17-20 isoprenoid units).

HO —CH2 —CH2 —C —CH2 + CH2 —CH = C —CH2 + CH2 —CH = C—CH

It should be noted that the first seven sugars (two GlcNAc and five Man residues) are donated by nucleotide sugars, whereas the last seven sugars (four Man and three Glc residues) added are donated by dolichol-P-sugars. The net result is assembly of the compound illustrated in Figure 47-8 and referred to in shorthand as Dol-P-P-GlcNAc2Man9Glc3.

The oligosaccharide linked to dolichol-P-P is transferred en bloc to form an N-glycosidic bond with one or more specific Asn residues of an acceptor protein emerging from the luminal surface of the membrane of the endoplasmic reticulum. The reaction is catalyzed by oligosaccharide:protein transferase, a membrane-associated enzyme complex. The transferase will recognize and transfer any substrate with the general structure Dol-P-P-(GlcNAc)2-R, but it has a strong preference for the Dol-P-P-GlcNAc2Man9Glc3 structure. Glycosylation occurs at the Asn residue of an Asn-X-Ser/Thr tripeptide sequence, where X is any amino acid except proline, aspartic acid, or glutamic acid. A tripeptide site contained within a P turn is favored. Only about one-third of the Asn residues that are potential acceptor sites are actually glycosylated, suggesting that factors other than the tripeptide are also important. The acceptor proteins are of both the secretory and integral membrane class. Cytosolic proteins are rarely glycosylated. The transfer reaction and subsequent processes in the glycosylation of N-linked glyco-proteins, along with their subcellular locations, are depicted in Figure 47-9. The other product of the oligosaccharide:protein transferase reaction is dolichol-P-P, which is subsequently converted to dolichol-P by a

UDP-GIcNAc

■ Tunicamycin

M — P — Dol and G — P — Dol (M)e — (GIcNAc)2 — P — P — Dol P — Dol

M — P — Dol ; M — (GIcNAc)2 — P — P — Dol

Figure 47-7. Pathway of biosynthesis of dolichol-P-P-oligosaccharide. The specific linkages formed are indicated in Figure 47-8. Note that the first five internal mannose residues are donated by GDP-mannose, whereas the more external mannose residues and the glucose residues are donated by dolichol-P-mannose and dolichol-P-glucose. (UDP, uridine diphosphate; Dol, dolichol; P, phosphate; UMP, uridine monophosphate; GDP, guanosine diphosphate; M, mannose; G, glucose.)

a1,2

Man-Man

^Man Man > GlcNAc > GlcNAc — P — P — Dolichol a1,2 a1,3 a1,3 a1,2 a1,2 /a1,3 Glc-Glc-Glc-Man-Man-Man

Figure 47-8. Structure of dolichol-P-P-oligosaccharide. (Reproduced, with permission, from Lennarz WJ: The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980.)

phosphatase. The dolichol-P can serve again as an acceptor for the synthesis of another molecule of Dol-P-P-oligosaccharide.

B. Processing of the Oligosaccharide Chain

1. Early phase—The various reactions involved are indicated in Figure 47-9. The oligosaccharide:protein transferase catalyzes reaction 1 (see above). Reactions 2 and 3 involve the removal of the terminal Glc residue by glucosidase I and of the next two Glc residues by glucosidase II, respectively. In the case of high-mannose glycoproteins, the process may stop here, or up to four Man residues may also be removed. However, to form complex chains, additional steps are necessary, as follows. Four external Man residues are removed in reactions 4 and 5 by at least two different mannosidases. In reaction 6, a GlcNAc residue is added to the Man residue of the Mana1-3 arm by GlcNAc transferase I. The action of this latter enzyme permits the occurrence of reaction 7, a reaction catalyzed by yet another mannosidase (Golgi a-mannosidase II) and which results in a reduction of the Man residues to the core number of three (Figure 47-5).

An important additional pathway is indicated in reactions I and II of Figure 47-9. This involves enzymes destined for lysosomes. Such enzymes are targeted to the lysosomes by a specific chemical marker. In reaction I, a residue of GlcNAc-1-P is added to carbon 6 of one or more specific Man residues of these enzymes. The reaction is catalyzed by a GlcNAc phosphotransferase, which uses UDP-GlcNAc as the donor and generates UMP as the other product:

GlcNAc PHOSPHOTRANSFERASE

In reaction II, the GlcNAc is removed by the action of a phosphodiesterase, leaving the Man residues phos-phorylated in the 6 position:

PHOSPHODIESTERASE

Man 6-P receptors, located in the Golgi apparatus, bind the Man 6-P residue of these enzymes and direct them to the lysosomes. Fibroblasts from patients with I-cell disease (see below) are severely deficient in the activity of the GlcNAc phosphotransferase. 2. Late phase—To assemble a typical complex oligosaccharide chain, additional sugars must be added to the structure formed in reaction 7. Hence, in reaction 8, a second GlcNAc is added to the peripheral Man residue of the other arm of the bi-antennary structure shown in Figure 47-9; the enzyme catalyzing this step is GlcNAc transferase II. Reactions 9, 10, and 11 involve the addition of Fuc, Gal, and NeuAc residues at the sites indicated, in reactions catalyzed by fucosyl, galactosyl, and sialyl transferases, respectively. The assembly of poly-A-acetyllactosamine chains requires additional GlcNAc transferases.

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