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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.


Mass spectral evidence for chitin-HA in HA made by SeHAS. Empty vector (a) or SeHAS (b) membranes were preincubated for 30 min with UDP-GlcNAc, UDP-GlcUA was added, and incubation proceeded for several minutes. The membranes were heated to release HA and subjected to two cycles of Folch extraction. Extracted HA products were speed vacuum concentrated, digested with ovine testicular hyaluronidase, and the resulting oligomers were subjected to affinity selection over carbograph. Eluted material was surveyed by MALDI-TOF MS to identify “hybrid” fragments corresponding to a chitin oligomer cap linked to HA disaccharides, such as the GlcNAc6-(GlcUA-GlcNAc)2 species shown.
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fig5: Mass spectral evidence for chitin-HA in HA made by SeHAS. Empty vector (a) or SeHAS (b) membranes were preincubated for 30 min with UDP-GlcNAc, UDP-GlcUA was added, and incubation proceeded for several minutes. The membranes were heated to release HA and subjected to two cycles of Folch extraction. Extracted HA products were speed vacuum concentrated, digested with ovine testicular hyaluronidase, and the resulting oligomers were subjected to affinity selection over carbograph. Eluted material was surveyed by MALDI-TOF MS to identify “hybrid” fragments corresponding to a chitin oligomer cap linked to HA disaccharides, such as the GlcNAc6-(GlcUA-GlcNAc)2 species shown.

Mentions: Although HAS does not require an exogenous primer to make HA, these novel self-made (GlcNAc)n-UDP products could potentially serve as endogenous primers that enable HAS to initiate HA chain assembly. In this proposed scenario (Figure 4), the nonreducing end of all HA chains retains this initial chitin oligomer primer, and all HA molecules have a novel non-HA structure (a chitin oligosaccharide cap) at their nonreducing end. Ongoing studies support this hypothesis [64], including HAS-dependent m/z signals indicating hybrid chitin-HA species such as GlcNAc6(GlcUA-GlcNAc)2 in ovine testicular hyaluronidase-digested samples (Figure 5(b)). Empty vector membranes without HAS (Figure 5(a)) or SeHAS membranes incubated without substrate (not shown) show no signals in this region. The hybrid chitin-HA digestion fragments can be affinity purified, fractionated by TLC or PAGE, and shown to contain multiple nonreducing GlcNAc residues that are releasable by treatment with jack bean hexosaminidase, as in Figure 3. Since we studied chitin-UDP oligomer products in vitro only, under conditions (e.g., exposure to a single sugar-UDP) that may not normally be encountered in cells, it remains to be demonstrated if these interesting products are also made in vivo. Studies are in progress to determine if HA molecules made by streptococcal and mammalian Class I HASs contain a nonreducing end chitin oligosaccharide cap. The presence of a chitin cap would have important physiologic implications for the polarity of HA chains and the potential ability of chitin-like binding proteins in the biomatrix or on cell surfaces to orient, align, and organize individual HA polymers into bundles; for example, cables or fibers [65].


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)

Mass spectral evidence for chitin-HA in HA made by SeHAS. Empty vector (a) or SeHAS (b) membranes were preincubated for 30 min with UDP-GlcNAc, UDP-GlcUA was added, and incubation proceeded for several minutes. The membranes were heated to release HA and subjected to two cycles of Folch extraction. Extracted HA products were speed vacuum concentrated, digested with ovine testicular hyaluronidase, and the resulting oligomers were subjected to affinity selection over carbograph. Eluted material was surveyed by MALDI-TOF MS to identify “hybrid” fragments corresponding to a chitin oligomer cap linked to HA disaccharides, such as the GlcNAc6-(GlcUA-GlcNAc)2 species shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581545&req=5

fig5: Mass spectral evidence for chitin-HA in HA made by SeHAS. Empty vector (a) or SeHAS (b) membranes were preincubated for 30 min with UDP-GlcNAc, UDP-GlcUA was added, and incubation proceeded for several minutes. The membranes were heated to release HA and subjected to two cycles of Folch extraction. Extracted HA products were speed vacuum concentrated, digested with ovine testicular hyaluronidase, and the resulting oligomers were subjected to affinity selection over carbograph. Eluted material was surveyed by MALDI-TOF MS to identify “hybrid” fragments corresponding to a chitin oligomer cap linked to HA disaccharides, such as the GlcNAc6-(GlcUA-GlcNAc)2 species shown.
Mentions: Although HAS does not require an exogenous primer to make HA, these novel self-made (GlcNAc)n-UDP products could potentially serve as endogenous primers that enable HAS to initiate HA chain assembly. In this proposed scenario (Figure 4), the nonreducing end of all HA chains retains this initial chitin oligomer primer, and all HA molecules have a novel non-HA structure (a chitin oligosaccharide cap) at their nonreducing end. Ongoing studies support this hypothesis [64], including HAS-dependent m/z signals indicating hybrid chitin-HA species such as GlcNAc6(GlcUA-GlcNAc)2 in ovine testicular hyaluronidase-digested samples (Figure 5(b)). Empty vector membranes without HAS (Figure 5(a)) or SeHAS membranes incubated without substrate (not shown) show no signals in this region. The hybrid chitin-HA digestion fragments can be affinity purified, fractionated by TLC or PAGE, and shown to contain multiple nonreducing GlcNAc residues that are releasable by treatment with jack bean hexosaminidase, as in Figure 3. Since we studied chitin-UDP oligomer products in vitro only, under conditions (e.g., exposure to a single sugar-UDP) that may not normally be encountered in cells, it remains to be demonstrated if these interesting products are also made in vivo. Studies are in progress to determine if HA molecules made by streptococcal and mammalian Class I HASs contain a nonreducing end chitin oligosaccharide cap. The presence of a chitin cap would have important physiologic implications for the polarity of HA chains and the potential ability of chitin-like binding proteins in the biomatrix or on cell surfaces to orient, align, and organize individual HA polymers into bundles; for example, cables or fibers [65].

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.