PNGase F

Last updated
Peptide:N-glycosidase F
PNGaseF Structure unknown-organism unknown-datasource.png
Identifiers
SymbolPNGase F
PDB 1PGS
UniProt Q9XBM8
Search for
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]

Related Research Articles

<span class="mw-page-title-main">Glycoprotein</span> Protein with oligosaccharide modifications

Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins are often glycosylated.

A congenital disorder of glycosylation is one of several rare inborn errors of metabolism in which glycosylation of a variety of tissue proteins and/or lipids is deficient or defective. Congenital disorders of glycosylation are sometimes known as CDG syndromes. They often cause serious, sometimes fatal, malfunction of several different organ systems in affected infants. The most common sub-type is PMM2-CDG where the genetic defect leads to the loss of phosphomannomutase 2 (PMM2), the enzyme responsible for the conversion of mannose-6-phosphate into mannose-1-phosphate

Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule in order to form a glycoconjugate. In biology, glycosylation usually refers to an enzyme-catalysed reaction, whereas glycation may refer to a non-enzymatic reaction.

An oligosaccharide is a saccharide polymer containing a small number of monosaccharides. Oligosaccharides can have many functions including cell recognition and cell adhesion.

The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched.

<span class="mw-page-title-main">Oligosaccharyltransferase</span>

Oligosaccharyltransferase or OST (EC 2.4.1.119) is a membrane protein complex that transfers a 14-sugar oligosaccharide from dolichol to nascent protein. It is a type of glycosyltransferase. The sugar Glc3Man9GlcNAc2 (where Glc=Glucose, Man=Mannose, and GlcNAc=N-acetylglucosamine) is attached to an asparagine (Asn) residue in the sequence Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline. This sequence is called a glycosylation sequon. The reaction catalyzed by OST is the central step in the N-linked glycosylation pathway.

The enzyme mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (endoglycosidase H) (EC 3.2.1.96) has systematic name glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-β-glucosaminohydrolase. It is a highly specific endoglycosidase which cleaves asparagine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins. It is used for research purposes to deglycosylate glycoproteins and to monitor intracellular protein trafficking through the secretory pathway.

<span class="mw-page-title-main">Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase</span>

In enzymology, a peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (EC 3.5.1.52) is an enzyme that catalyzes a chemical reaction that cleaves a N4-(acetyl-beta-D-glucosaminyl)asparagine residue in which the glucosamine residue may be further glycosylated, to yield a (substituted) N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue. This enzyme belongs to the family of hydrolases, specifically those acting on carbon-nitrogen bonds other than peptide bonds in linear amides.

<span class="mw-page-title-main">MAN1B1</span> Protein-coding gene in the species Homo sapiens

Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase is an enzyme that in humans is encoded by the MAN1B1 gene.

<span class="mw-page-title-main">NGLY1</span> Protein-coding gene in the species Homo sapiens

PNGase also known as N-glycanase 1 or peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase is an enzyme that in humans is encoded by the NGLY1 gene. PNGase is a de-N-glycosylating enzyme that removes N-linked or asparagine-linked glycans (N-glycans) from glycoproteins. More specifically, NGLY1 catalyzes the hydrolysis of the amide bond between the innermost N-acetylglucosamine (GlcNAc) and an Asn residue on an N-glycoprotein, generating a de-N-glycosylated protein, in which the N-glycoylated Asn residue is converted to asp, and a 1-amino-GlcNAc-containing free oligosaccharide. Ammonia is then spontaneously released from the 1-amino GlcNAc at physiological pH (<8), giving rise to a free oligosaccharide with an N,N’-diacetylchitobiose structure at the reducing end.

<span class="mw-page-title-main">MAN2A1</span> Protein-coding gene in the species Homo sapiens

Alpha-mannosidase 2 is an enzyme that in humans is encoded by the MAN2A1 gene.

<span class="mw-page-title-main">MGAT2</span> Protein-coding gene in the species Homo sapiens

Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase is an enzyme that in humans is encoded by the MGAT2 gene.

<span class="mw-page-title-main">MAN2B2</span> Protein-coding gene in the species Homo sapiens

Epididymis-specific alpha-mannosidase is an enzyme that in humans is encoded by the MAN2B2 gene.

<span class="mw-page-title-main">MAN1A1</span> Protein-coding gene in the species Homo sapiens

Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA is an enzyme that in humans is encoded by the MAN1A1 gene.

<span class="mw-page-title-main">MGAT5B</span> Protein-coding gene in the species Homo sapiens

Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B is an enzyme that in humans is encoded by the MGAT5B gene.

Glycopeptides are peptides that contain carbohydrate moieties (glycans) covalently attached to the side chains of the amino acid residues that constitute the peptide.

<i>N</i>-linked glycosylation Attachment of an oligosaccharide to a nitrogen atom

N-linked glycosylation, is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom, in a process called N-glycosylation, studied in biochemistry. The resulting protein is called an N-linked glycan, or simply an N-glycan.

Mannosyl-oligosaccharide glucosidase (MOGS) (EC 3.2.1.106, processing α-glucosidase I,Glc3Man9NAc2 oligosaccharide glucosidase, trimming glucosidase I, GCS1) is an enzyme with systematic name mannosyl-oligosaccharide glucohydrolase. MOGS is a transmembrane protein found in the membrane of the endoplasmic reticulum of eukaryotic cells. Biologically, it functions within the N-glycosylation pathway.

<span class="mw-page-title-main">Paucimannosylation</span> Protein Paucimannosylation

In biochemistry, paucimannosylation is an enzymatic post-translational modification involving the attachment of relatively simple mannose (Man) and N-Acetylglucosamine (GlcNAc) containing carbohydrates (glycans) to proteins. The paucimannosidic glycans may also be modified with other types of monosaccharides including fucose (Fuc) and xylose (Xyl) depending on the species, tissue and cell origin.

<span class="mw-page-title-main">Glycan-protein interactions</span> Class of biological intermolecular interactions

Glycan-Protein interactions represent a class of biomolecular interactions that occur between free or protein-bound glycans and their cognate binding partners. Intramolecular glycan-protein (protein-glycan) interactions occur between glycans and proteins that they are covalently attached to. Together with protein-protein interactions, they form a mechanistic basis for many essential cell processes, especially for cell-cell interactions and host-cell interactions. For instance, SARS-CoV-2, the causative agent of COVID-19, employs its extensively glycosylated spike (S) protein to bind to the ACE2 receptor, allowing it to enter host cells. The spike protein is a trimeric structure, with each subunit containing 22 N-glycosylation sites, making it an attractive target for vaccine search.

References

  1. 1 2 3 4 5 Tarentino AL, Trimble RB, Plummer TH (1989). Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. 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.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.