Role of <i>N</i>‐linked oligosaccharides attached to human renin expressed in COS cells

Hori, Hitoshi; Yoshino, Teruhiko; Ishizuka, Yasuyuki; Yamauchi, Takeshi; Murakami, Kazuo

doi:10.1016/0014-5793(88)80777-4

Cited by 19 publications

(6 citation statements)

References 15 publications

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“…4A), and the conserved presence of a well defined glycosylation site in a similar region of these three aspartic proteinases might indicate a common biological role. It is possible that they protect these hydrolases against accelerated proteolytic cleavage and therefore play a role in the stability of the enzymes, similar to what was found for recombinant renin expressed in COS cells (47) or for Mucor aspartic proteinase expressed in yeast (48).…”

Section: Resultsmentioning

confidence: 84%

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers ofCynara cardunculus L.

Frazão¹,

Bento²,

Costa³

et al. 1999

Journal of Biological Chemistry

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Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Man␤4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two ␤-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed. Aspartic proteinases (AP)1 are a class of enzymes (EC 3.4.23) involved in a number of physiological and pathological processes such as blood pressure homeostasis (renin), retroviral infection (human immunodeficiency virus proteinase), hemoglobin degradation in malaria (plasmepsin), intracellular proteolysis (cathepsin D),and digestion (pepsin) (see Ref. 1 for a recent review). Additionally, AP play an important role in the food industry, e.g. the cheese industry (chymosin) or in soya and cocoa processing. Inhibitors of AP enzymes have significant therapeutic potential for treatment of hypertension, AIDS, tumor invasiveness, and peptic ulcer disease. Despite their distribution in the living world, occurring from retrovirus to mammals, aspartic proteinases share significant similarities in primary and tertiary structures. Members of this class display two Asp-Thr/Ser-Gly motifs within their sequences and are specifically inhibited by pepstatin, a peptide produced by Streptomyces. Several high resolution x-ray structures of mammalian, fungal, and retroviral aspartic proteinases are available. The overall three-dimensional structure consists of two domains of similar secondary structure, dominated by orthogonally packed sheets with several small helical segments. In eukaryotic aspartic proteinases each domain contributes one of the two catalytic aspartate residues to form the active site center located at a long and deep cleft between the two domains. Conversely, in retroviral aspartic proteinases, which are dimeric proteins consisting of two identical subunits, each subunit contributes one catalytic aspartate. It is thought therefore that eukaryotic aspartic proteinases have evolved divergently from a primitive dimeric enzyme resembling retroviral proteinases by gene duplication and fusion.Plant AP have been detected, extracted...

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Section: Resultsmentioning

confidence: 84%

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers ofCynara cardunculus L.

Frazão¹,

Bento²,

Costa³

et al. 1999

Journal of Biological Chemistry

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“…Thus, it appears that N‐glycosylation does not play an essential role for the activity of the plant aspartic proteinases as the sites of addition of N‐glycans are not conserved in the different proteins. Glycosylation of human renin expressed in monkey cells improved the stability of the protein [33] suggesting another potential function of this modification in the plant enzymes. Thus, we obtained protein sequence from the aspartic proteinase purified from seeds [21] that showed that this enzyme was derived from the AtPasp A1 and A2 genes.…”

Section: The Seed Proteinase Is Derived From Two Of These Genesmentioning

confidence: 99%

The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed

Xia

Pfeil

Gal

2002

European Journal of Biochemistry

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Genomic sequencing has identified three different typical plant aspartic proteinases in the genome of Arabidopsis thaliana, named Pasp-A1, A2 and A3. A1 is identical to a cDNA we had previously isolated and the two others produce proteins 81 and 63% identical to that predicted protein.Sequencing of the aspartic proteinase protein purified from Arabidopsis seeds showed that the peptides are derived from two of these genes, A1 and A2. Using gene specific probes, we have analyzed RNA from different tissues and found these three genes are differentially expressed. A1 mRNA is detected in all tissues analyzed and more abundant in leaves during the light phase of growth. The other two genes are expressed either primarily in flowers (A3) or in seeds (A2).In situ hybridization demonstrated that all three genes are expressed in many cells of the seeds and developing seed pods. The A1 and A3 genes are expressed in the sepals and petals of flowers as well as the outer layer of the style, but are not expressed in the transmitting tract or on the stigmatal surface. The A2 gene is weakly expressed only in the transmitting tissue of the style. All three genes are also expressed in the guard cells of sepals. These data suggest multiple roles for aspartic proteinases besides those proposed in seeds.

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“…We have recently reported that the glycosylation-deficient human renin (NA575) is unstable m monkey kidney COS cells transfected with its expression plasmid [25]. In this study, we demonstrated that glycosylation of renin plays a pivotal role in its intracellular sorting in oocytes, probably due to its phosphorylation by UDP-GlcNAc: lysosomal enzyme N-acetylglucosaminylphosphotransferase.…”

Section: Discussionmentioning

confidence: 76%

“…Site-directed mutagenesis to substitute the asparagine residues of glycosylation sites of human renin was performed as described previously [25]. The mutated cDNA fragment was exchanged with the corresponding one of the plasmid of native human preprorenin.…”

Section: Methodsmentioning

confidence: 99%

The influence of glycosylation on the fate of renin expressed in Xenopus oocytes

Nakayama

Hatsuzawa

Kim

et al. 1990

European Journal of Biochemistry

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It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine-linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Renl renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Renl renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Renl renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino-acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renin remains intracellular if it is only glycosylated at one of the two sites.

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Role of N‐linked oligosaccharides attached to human renin expressed in COS cells

Cited by 19 publications

References 15 publications

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers ofCynara cardunculus L.

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers ofCynara cardunculus L.

The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed

The influence of glycosylation on the fate of renin expressed in Xenopus oocytes

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