PNGase F

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Peptide:N-glycosidase F
PNGaseF Structure unknown-organism unknown-datasource.png
Identifiers
SymbolPNGase F
PDB 1PGS
UniProt Q9XBM8
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Structures Swiss-model
Domains InterPro

Peptide:N-glycosidase F, commonly referred to as PNGase F, is an amidase of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase class. PNGase F works by cleaving between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides. This results in a deaminated protein or peptide and a free glycan. [1] [2]

Contents

PNGase F has a molecular weight of 35,500 and consists of a polypeptide chain of 314 amino acids. [3] The optimal pH for enzyme activity is 8.6. However, the activity is stable for a wide variety of conditions and reagents. PNGase F maintains 60% activity from pH 6.0 to pH 9.5. It is able to deglycosylate in the absence of denaturants, but needs extensive incubation and larger amounts of the enzyme to cleave native proteins. [1] [4] [5]

Other endoglycosidases, similar to PNGase F, include endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. [6] [7] [8] These endoglycosidases have more specificity in cleavage and are less sensitive to protein conformation than PNGase F. [1] [9] [10] All of these endoglycosidases, including PNGase F, can be purified from an almond emulsion or flavobacterium meningosepticum. [1] [6] [10] [11] [12]

Mechanism

PNGase F catalyzes the cleavage of an internal glycoside bond in an oligosaccharide. It cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless the core GlcNAc contains an alpha 1,3- fucose. [1]

PNGase F cleavage sites. PNGase F Specificity.png
PNGase F cleavage sites.

The asparagine residue, from which the glycan is removed, is deaminated to aspartic acid.

PNGase F cleaves glycan and deaminates asparagine to aspartic acid. PNGaseF Mechanism.png
PNGase F cleaves glycan and deaminates asparagine to aspartic acid.

PNGase F requires a minimum of two GlcNAc oligosaccharide residues attached to the asparagine in order for catalysis to occur. [12] This enzyme utilizes a catalytic triad of cysteine-histidine-aspartate in its active site, which is a common motif for amidases. This motif contains a nucleophile, a proton donor, and a positive charge to stabilize the tetrahedral intermediate. The crystal structure of PNGase F from flavobacterium miningosepticum, with 1.8 Å resolution, was found to be folded in two domains, each with an eight-stranded antiparallel β barrel, or jelly roll, configuration. This structure is similar to lectins and glucanases, suggesting similarities with lectins and other carbohydrate-binding proteins. [3]

Applications and uses

Biologically, deficiencies in endoglycosidases can lead to several diseases, including lysosomal storage diseases and multisystem diseases, most of which involve the nervous system. [13] [14] N-linked glycans can provide structural components of cell walls and extracellular matrices, modify protein stability and solubility, direct trafficking of other glycoproteins, and mediate cell signaling (cell-cell interactions and cell-matrix interactions). [15] N-linked glycosylation can be seen in antibodies, on cell surfaces, and on various proteins throughout the matrix. Alterations in glycosylation are often acquired in cases of cancer and inflammation, which may have important functional consequences. [16]

To that end, PNGase F and other endoglycosidases can be used to study oligosaccharides and characterize glycoproteins. PNGase F lacks selectivity for outer carbohydrate structure, resulting in broad specificity, making it a useful tool for investigating glycoprotein structure and function. [3] In most instances, proteins of interest are denatured and treated with PNGase F. Following this, they are either subjected to gel electrophoresis, in which protein migration changes due to the deglycosylation by PNGase F, or are analyzed via mass spectrometry, by which the oligosaccharide can be characterized and the protein or peptide fragment from which it came can be characterized. [3] [7] [8]

References

  1. 1 2 3 4 5 Tarentino AL, Trimble RB, Plummer TH (1989). "Chapter 5 Enzymatic Approaches for Studying the Structure, Synthesis, and Processing of Glycoproteins". Vesicular Transport Part B. Methods in Cell Biology. Vol. 32. pp. 111–39. doi:10.1016/S0091-679X(08)61169-3. ISBN   978-0-08-085930-9. PMID   2691848.
  2. ↑ Tarentino AL, Plummer TH (1994). "[4] Enzymatic deglycosylation of asparagine-linked glycans: Purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum". Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology. Vol. 230. pp. 44–57. doi:10.1016/0076-6879(94)30006-2. ISBN   9780121821319. PMID   8139511.
  3. 1 2 3 4 Norris GE, Stillman TJ, Anderson BF, Baker EN (1994). "The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum". Structure. 2 (11): 1049–59. doi: 10.1016/S0969-2126(94)00108-1 . PMID   7881905.
  4. ↑ Anthony L., Tarentino and Thomas H. Plummer Jr.. "Enzymatic deglycosylation of asparagine-linked glycans: Purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum." Methods in Enzymology. 230. 1994. 44-57. Web.
  5. ↑ Tarentino AL, Plummer TH (1982). "Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding reagents". The Journal of Biological Chemistry. 257 (18): 10776–80. doi: 10.1016/S0021-9258(18)33891-2 . PMID   7107633.
  6. 1 2 Takahashi T, Nishibe H (1981). "Almond glycopeptidase acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity". Biochimica et Biophysica Acta (BBA) - Enzymology. 657 (2): 457–67. doi:10.1016/0005-2744(81)90331-4. PMID   7213757.
  7. 1 2 Maley F, Trimble RB, Tarentino AL, Plummer TH (1989). "Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases". Analytical Biochemistry. 180 (2): 195–204. doi:10.1016/0003-2697(89)90115-2. PMID   2510544.
  8. 1 2 Tachibana Y, Yamashita K, Kobata A (1982). "Substrate specificity of mammalian endo-beta-N-acetylglucosaminidase: study with the enzyme of rat liver". Archives of Biochemistry and Biophysics. 214 (1): 199–210. doi:10.1016/0003-9861(82)90023-6. PMID   6805439.
  9. ↑ Taga EM, Waheed A, Van Etten RL (1984). "Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond". Biochemistry. 23 (5): 815–22. doi:10.1021/bi00300a006. PMID   6712926.
  10. 1 2 Tarentino AL, Gómez CM, Plummer TH (1985). "Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F". Biochemistry. 24 (17): 4665–71. doi:10.1021/bi00338a028. PMID   4063349.
  11. ↑ Plummer TH, Tarentino AL (1981). "Facile cleavage of complex oligosaccharides from glycopeptides by almond emulsin peptide: N-glycosidase" (PDF). The Journal of Biological Chemistry. 256 (20): 10243–6. doi: 10.1016/S0021-9258(19)68610-2 . PMID   7287707.
  12. 1 2 Plummer TH, Phelan AW, Tarentino AL (1987). "Detection and quantification of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidases". European Journal of Biochemistry. 163 (1): 167–73. doi: 10.1111/j.1432-1033.1987.tb10751.x . PMID   2434326.
  13. ↑ Davies G, Henrissat B (1995). "Structures and mechanisms of glycosyl hydrolases". Structure. 3 (9): 853–9. doi: 10.1016/S0969-2126(01)00220-9 . PMID   8535779.
  14. ↑ Patterson MC (2005). "Metabolic mimics: the disorders of N-linked glycosylation". Seminars in Pediatric Neurology. 12 (3): 144–51. doi:10.1016/j.spen.2005.10.002. PMID   16584073.
  15. ↑ Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, Varki A, Sharon N (2009). "Historical Background and Overview". In Varki A (ed.). Essentials of Glycobiology (2nd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN   978-0-87969-770-9. PMID   20301255.
  16. ↑ Rhodes J, Campbell BJ, Yu LG (2001). "Glycosylation and Disease". Encyclopedia of Life Sciences. John Wiley & Sons, Inc. doi:10.1002/9780470015902.a0002151.pub2. ISBN   978-0-470-01590-2.
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