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α1-Antitrypsin: Structure, Function, Physiology
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
The predominant site of synthesis of plasma α1-AT is liver. This is most clearly shown by conversion of plasma α1-AT to donor phenotype after orthotopic liver transplantation.7,8 It is synthesized in human hepatoma cells as a 52-kDa precursor, undergoes posttranslational dolichol phosphate-linked glycosylation at three asparagine residues, and also undergoes tyrosine sulfation.61,62 It is secreted as a 55-kDa native single-chain glycoprotein with a halftime for secretion of 35 to 40 min.
Molecular Structure and Functions of Collagen
Published in Marcel E. Nimni, Collagen, 1988
Marcel E. Nimni, Robert D. Harkness
As lysyl residues in the newly synthesized proa chains are hydroxylated, sugar residues are added to the resulting hydroxylysyl groups. Glycosylations are catalyzed by two specific enzymes, a galactosyltransferase and a glucosyltransferase.36 The first of these enzymes adds galactose to the hydroxylysyl residues, and the second adds glucose to the galactosylhydroxylsine that is formed. The galactosyltransferase from chick embryo has been purified about 1000-fold and the glucosyltransferase has been isolated as a homogeneous protein. Both enzymes are glycoproteins and their activity requires the presence of sulfhydryl groups. The activity of partially purified galactosyltransferase is separated by gel filtration into three species with apparent molecular weights of 450,000, 200,000, and 50,000 daltons. The purified glucosyltransferase has a molecular weight of 70,000 daltons. Both these transferases use sugar in a form of a uridine diphosphate glycoside, and require the presence of bivalent cations, preferably manganese.37 These enzymes, like the hydroxylases, require that the proa chains be in a nonhelical conformation. In intact cells, glycosylation is initiated while the polypeptides are still being assembled on the ribosomes, but probably continues after the release of complete proa chains in the cisternae of the rough ER; activity ceases when the chains acquire a triple-helical conformation. The oligosaccharides present in the extension peptides associated with the C-terminal region of collagen resemble those present in most other glycoproteins; they contain N-acetylglucosamine and mannose and are attached to asparagine residues.38,39 Their composition suggests that they are added as intermediates via the dolichol phosphate pathway and that final remodeling occurs in the Golgi after the helix has been formed.40 Once the translation, modifications, and additions are completed, it is essential that the individual proa chains become properly aligned for the triple helix to form. We do not know if this alignment occurs while the polypeptides are still attached to the ribosome or if they have to detach, or if the N-terminal “signal” peptide plays a role in this connection. In any case, proper alignment should juxtapose the appropriate cysteine residues as a prerequisite for formation of the disulfide bridges that link the individual proa chains at the C-terminal end. Earlier studies involving subcellular fractionation suggested that the disulfide bridges could appear during translocation of procollagen from the ribosome to the smooth endoplasmic reticulum, probably in the cisternae of the ER.41 More recently, it has been proposed that disulfide bond formation occurs while the propeptides are still attached to the ribosome.42 In any case, it seems clear that for assembly and secretion the C-terminal extensions must be present.43–45
Targeting on glycosylation of mutant FLT3 in acute myeloid leukemia
Published in Hematology, 2019
Tunicamycin(TM) is a bacterial antibiotic that significantly inhibits the transfer of active sugars to dolichol phosphate, which is an important step in the N-glycosylation of protein at endoplasmic reticulum [46,47]. TM inhibits the synthesis of N-linked oligosaccharides in many cells [72]. The expression of membrane receptors that show carcinogenicity can be decreased by TM [73]. Downstream signaling pathways were interrupted by N-linked glycosylation suggesting that TM may be an alternative treatment to reduce carcinogenic signals and drug resistance [74]. TM exhibited cytotoxic effects and enhance the susceptibility of therapy on different cell lines including human head and neck carcinoma and lung cancer cells through glycosylation inhibition [75]. In lung cancer cells, deglycosylation of human pentraxin-3 (PTX3) protein by TM enhanced the sensitivity to therapy via AKT/NF-κB signaling pathway. TM inhibits glycosylation of plasma membrane receptors, resulting in impaired transport of these receptors to the cell surface [76,77].
Mass spectrometry for the identification and analysis of highly complex glycosylation of therapeutic or pathogenic proteins
Published in Expert Review of Proteomics, 2020
Yukako Ohyama, Kazuki Nakajima, Matthew B. Renfrow, Jan Novak, Kazuo Takahashi
N-glycan diversity is created during the process of N-glycosylation. N-glycosylation starts from the formation of a glyco-lipid precursor. A branched carbohydrate structure consisting of glucose (Glc)-containing glycans, (Glc)3(Man)9GlcNAc2, is attached to dolichol phosphate that is then ‘flipped’ into the lumen of the endoplasmic reticulum (ER) (Figure 1(a)). An oligosaccharyltransferase adds the carbohydrate chain to the Asn residue of the Asn-X-Thr/Ser consensus sequence of the nascent protein. After further removal of the Glc residues in the ER, the quality-control step, the folded glycoprotein moves to the cis face of the Golgi apparatus for additional removal of Man by α-mannosidase I (α-Man I; MAN1A1, MAN1A2, MAN1C1) to form Man5GlcNAc2 [2,3,6]. Further modifications are performed by GlcNAc-transferase (GnT)-I to form GlcNAcMan5GlcNAc2. Subsequently, the majority of N-glycans are trimmed by α-mannosidase II (α-Man II; MAN2A1, MAN2A2) to form GlcNAcMan3GlcNAc2 in the medial-Golgi. Once both Man residues are removed, a second GlcNAc is added to the C-2 of the α1-6 Man in the N-glycan core by the action of GnT-II to yield the precursor for all biantennary complex N-glycans; GlcNAc2Man3GlcNAc2. Additional branches can be added at C-4 of the core α1-3 Man and at C-6 of the core α1-6 Man by GnT-IV (MGAT4A, MGAT4B) and GnT-V (MGAT5, MGAT5B). The ‘bisecting’ GlcNAc residue can be attached to the β-Man of the core by GnT-III (MGAT3). Hybrid N-glycans are formed if the GlcNAcMan5GlcNAc2 glycan produced by GnT-I is not digested by α-Man II (MAN2A1,MAN2A2) [6]. Further modifications are performed by α1-6-fucosyltransferase (FUT8), β1,4-galactosyltransferases (Gal-T), α2,3-sialyltransferase (α2,3 Sialyl-T) and α2,6-sialyltransferase (α2,6 Sialyl-T), which is distributed in the medial to trans face of Golgi apparatus [3].
Comprehensive manipulation of glycosylation profiles across development scales
Published in mAbs, 2019
Sven Loebrich, Elisa Clark, Kristina Ladd, Stefani Takahashi, Anna Brousseau, Seth Kitchener, Robert Herbst, Thomas Ryll
Numerous media additives have been used to modulate glycosylation structures, including galactose, glucosamine,15,19 N-acetyl glucosamine (GlucNAc), uridine, mannose, N-acetyl neuraminic acid (NeuNAc),16 N-acetyl mannosamine (ManNAc),17,18 ammonia,19 manganese,20 dolichol phosphate,21 cytidine,22 and glycerol.23,36 Similarly, a variety of enzyme inhibitors has been exploited.24–27 In our study, we tested 10 commonly used media additives and characterized their effect on the glycosylation profile of different IgG1 molecules. We found that feeding of 12.5 mM glucosamine led to dramatic decreases in galactosylated species, in line with observations by others.16,37 For example, Hills and colleagues saw a 57% drop in galactosylation in GS-NS0 cells in response to 10 mM glucosamine.15 The same concentration has also been reported to reduce the degree of sialylation,17,19 (reviewed in ref. 1), in line with the notion that the addition of sialic acid residues occurs on terminally galactosylated glycoforms (reviewed in refs. 1,10, and 11). The suppression of galactosylated species in response to glucosamine feeding is widely believed to stem from competition for UTP in the formation of UDP-GlcNAc, which hampers generation of UDP-Gal.38 In addition, competitive inhibition of the UDP-galactose transporter may play a role. Both mechanisms result in limited availability of activated galactose in the lumen of the Golgi cisternae. We observed a mild retardation of cell growth in response to glucosamine feeding, a phenomenon that has been described by others 37 and is believed to result either from depletion of the cytosolic acetyl-CoA pool as a consequence of the acetylation of glucosamine to GlcNAc,37 or from inhibition of glucose uptake.19 In addition, we observed an increase in Man5 species in response to glucosamine supplementation. Others have reported a decrease in high mannose species after glucosamine feeding, albeit in Madin-Darby canine kidney cells,39 and in NS0 cells15 (reviewed in ref 10).