Notes

Type Iii
Fucosylated Lactose calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins.Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if Oligosaccharides is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Gl-3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding.

To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12.Escherichia coli K-12 harboring a part of the structural genes for the Klebsiella K2 capsular polysaccharide (cpsK) expresses a large amount of K2 capsular polysaccharide as a thick capsule in the presence of plasmids carrying rmpA and rcsB.

We have previously shown that expression of the Klebsiella K2 capsule in E. coli HB1 harboring cpsK depends on the presence of rmpA, a regulatory gene from a large plasmid of Klebsiella pneumoniae Chedid (O1K2). E. coli K-12 JM9, however, produces only a small amount of K2 capsular polysaccharide, even in the presence of plasmids carrying rmpA as well as the cpsK structural genes. Introduction of the rcsB gene, a positive regulator of colanic acid capsule synthesis in E. coli K-12 which was cloned from HB1 on a plasmid, into JM9 cells carrying cpsK and rmpA, results in the expression of a thick K2 capsule. By Northern (RNA) hybridization analysis, rcsB has been found to enhance transcription of a long strand of mRNA (longer than 14 kb) from cpsK.

These E. coli transformants which produce a thick K2 capsule also express colanic acid production at high levels. Therefore, rcsB can act as a positive regulator of Klebsiella K2 capsule production and two capsular polysaccharides can be expressed in E. coli simultaneously. With a somewhat different strain background, we have found that both of the colanic acid regulators, rcsA and rcsB, contribute to the basal level of Klebsiella K2 capsule expression but that the presence of multicopy rcsB in either an rcsB or an rcsA mutant of E. coli is sufficient to increase the expression of K2 capsular polysaccharide. These results suggest further parallels between the regulation of colanic acid synthesis in E.

coli and the regulation of Klebsiella K2 capsule synthesis.Evidence for covalent attachment of phospholipid to the capsular polysaccharide Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent.
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