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Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.


Glycosidase treatment converts larger (GlcNAc)n-UDP oligomers to GlcNAc-UDP. SeHAS membranes were incubated for 30 min with UDP-GlcNAc alone, Folch extracted, fractionated over a size exclusion column, and samples were either untreated (0 min) or treated (120 min) with jack bean hexosaminidase. The samples were then analyzed by MALDI-TOF MS to identify and quantify m/z signals of candidate oligomeric chitin-UDP fragments corresponding to n = 1–4 (boldface white or black numbers). The presence of chitin linkages was confirmed by the ability of glycosidase treatment to shift species with 3 or 4 sugars to products with 1 (GlcNAc-UDP) or 2 sugars. Additional MS/MS analysis of the starting sample ions (not shown) revealed smaller members of the expected oligomer series, including GlcNAc-UDP.
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fig3: Glycosidase treatment converts larger (GlcNAc)n-UDP oligomers to GlcNAc-UDP. SeHAS membranes were incubated for 30 min with UDP-GlcNAc alone, Folch extracted, fractionated over a size exclusion column, and samples were either untreated (0 min) or treated (120 min) with jack bean hexosaminidase. The samples were then analyzed by MALDI-TOF MS to identify and quantify m/z signals of candidate oligomeric chitin-UDP fragments corresponding to n = 1–4 (boldface white or black numbers). The presence of chitin linkages was confirmed by the ability of glycosidase treatment to shift species with 3 or 4 sugars to products with 1 (GlcNAc-UDP) or 2 sugars. Additional MS/MS analysis of the starting sample ions (not shown) revealed smaller members of the expected oligomer series, including GlcNAc-UDP.

Mentions: SeHAS synthesizes chitin oligomers, (GlcNAc-β1,4)n [61], as reported for XlHAS1 [62] and MmHAS1 [63]. More importantly, however, and consistent with reducing end sugar addition, we found that SeHAS also makes novel chitin oligomers attached to -GlcNAc(α1→)UDP at the reducing end [61]. SeHAS incubated with only GlcNAc-UDP makes a series of (GlcNAc-β1,4)n-GlcNAc(α1→)UDP oligomers (for n = 2–15) corresponding to (GlcNAc)2-UDP through (GlcNAc)7-UDP products. SeHAS membranes incubated without substrate or with only GlcUA-UDP show no signals in this region. Product identifications were confirmed by MS-MS fragmentation and digestion with jack bean β-N-acetylglucosaminidase (e.g., all species ultimately yielded GlcNAc-UDP). For example, tri- and tetraoligomers were confirmed to contain β1,4-linked GlcNAc residues attached to GlcNAc-UDP because treatment with jack bean hexosaminidase converted almost all the initial oligomers to GlcNAc-UDP or (GlcNAc)2-UDP (Figure 3). Thus, HAS synthesizes (GlcNAc-β1,4)1–7-GlcNAc(α1→)UDP oligomers. These unusual sugar-nucleotide species, activated by α-attachment to UDP, are unstable and readily cleaved to yield chitin oligomers, explaining the ability of Class I HASs to make chitin.


Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Glycosidase treatment converts larger (GlcNAc)n-UDP oligomers to GlcNAc-UDP. SeHAS membranes were incubated for 30 min with UDP-GlcNAc alone, Folch extracted, fractionated over a size exclusion column, and samples were either untreated (0 min) or treated (120 min) with jack bean hexosaminidase. The samples were then analyzed by MALDI-TOF MS to identify and quantify m/z signals of candidate oligomeric chitin-UDP fragments corresponding to n = 1–4 (boldface white or black numbers). The presence of chitin linkages was confirmed by the ability of glycosidase treatment to shift species with 3 or 4 sugars to products with 1 (GlcNAc-UDP) or 2 sugars. Additional MS/MS analysis of the starting sample ions (not shown) revealed smaller members of the expected oligomer series, including GlcNAc-UDP.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4581545&req=5

fig3: Glycosidase treatment converts larger (GlcNAc)n-UDP oligomers to GlcNAc-UDP. SeHAS membranes were incubated for 30 min with UDP-GlcNAc alone, Folch extracted, fractionated over a size exclusion column, and samples were either untreated (0 min) or treated (120 min) with jack bean hexosaminidase. The samples were then analyzed by MALDI-TOF MS to identify and quantify m/z signals of candidate oligomeric chitin-UDP fragments corresponding to n = 1–4 (boldface white or black numbers). The presence of chitin linkages was confirmed by the ability of glycosidase treatment to shift species with 3 or 4 sugars to products with 1 (GlcNAc-UDP) or 2 sugars. Additional MS/MS analysis of the starting sample ions (not shown) revealed smaller members of the expected oligomer series, including GlcNAc-UDP.
Mentions: SeHAS synthesizes chitin oligomers, (GlcNAc-β1,4)n [61], as reported for XlHAS1 [62] and MmHAS1 [63]. More importantly, however, and consistent with reducing end sugar addition, we found that SeHAS also makes novel chitin oligomers attached to -GlcNAc(α1→)UDP at the reducing end [61]. SeHAS incubated with only GlcNAc-UDP makes a series of (GlcNAc-β1,4)n-GlcNAc(α1→)UDP oligomers (for n = 2–15) corresponding to (GlcNAc)2-UDP through (GlcNAc)7-UDP products. SeHAS membranes incubated without substrate or with only GlcUA-UDP show no signals in this region. Product identifications were confirmed by MS-MS fragmentation and digestion with jack bean β-N-acetylglucosaminidase (e.g., all species ultimately yielded GlcNAc-UDP). For example, tri- and tetraoligomers were confirmed to contain β1,4-linked GlcNAc residues attached to GlcNAc-UDP because treatment with jack bean hexosaminidase converted almost all the initial oligomers to GlcNAc-UDP or (GlcNAc)2-UDP (Figure 3). Thus, HAS synthesizes (GlcNAc-β1,4)1–7-GlcNAc(α1→)UDP oligomers. These unusual sugar-nucleotide species, activated by α-attachment to UDP, are unstable and readily cleaved to yield chitin oligomers, explaining the ability of Class I HASs to make chitin.

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.