Protein Glycosylation in Yeast

An efficient expressiordpurification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUCZ). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI.Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidasedeficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98 % pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecularmass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting K,-values of ~1 . 5 nM and ~1 . 6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of ~0 . 1 nM and = I nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.

Section: Discussionmentioning

confidence: 99%

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

Pohlig

Fendrich

Knecht

et al. 1996

“…4). The core oligosaccharide of N-linked glycoproteins is extended in the yeast Golgi apparatus by addition of a1,6-linked, al,2-linked and al,3-linked mannose residues (for review see: Kukuruzinska et al, 1987;Tanner and Lehle, 1987). For testing the specifity of the man-al,6-man residues, lysates of a yeast strain transformed with pDP34/ GAPFL-PHOS were also immunoprecipitated with a specific antibody to acid phosphatase.…”

Section: Retention Of St In the Er Of Yeastmentioning

confidence: 99%

“…To examine biosynthesis and intracellular transport of heterologously expressed human enzymes, we took advantage of the fact that both enzymes are glycoproteins and carry one (GT) or two (ST) N-linked and various 0-linked glycan chains, Whereas initial steps of N-glycosylation of proteins are conserved in eucaryotic cells, 0-glycosylation in S. cerevisiae differs from that in higher eucaryotic cells (for review see Kukuruzinska et al, 1987;Tanner and Lehle, 1987): Briefly, the pathway for the synthesis of N-linked glycans in yeast is, in early steps, very similar to that of higher eucaryotic cells. First, the co-translational transfer of a core oligosaccharide (Man,GlcNAc,) from dolichol pyrophosphate to the amide group of Asp in an Asn-Xaa-Thr(Ser) sequon of the host protein takes place on the luminal side of the endoplasmic reticulum membrane.…”

mentioning

confidence: 99%

Human β1,4 galactosyltransferase and α2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum

Krezdorn¹,

Kleene²,

Watzele³

et al. 1994

Biosynthesis and intracellular transport of recombinant human full-length Pl,4 galactosyltransferase (GT) and full-length a2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C . , Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur: J. Biochem. 212, 113-1201. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 'H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of P-galactosidase-GT or P-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radioactive sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-al,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus.These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

“…The difference of 162 Da between each of the multiple forms is characteristic of one hexose residue. The sugar-chain heterogeneity is probably due to incomplete sugar processing during post-translational modification, and the hexose is likely to be mannose [24]. Based on the relative intensities for each molecular mass, it was esti- Tilapla LLLGAFNIKSFGDTKASNATLMNIITKIVKRYDVILIQEVRDSDLSATQTLMNYVNKDSP-QYKYIVSEPLGASTYKERYLFLYRE4LVSWKSYTYDDG 99 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 c1 c2 c3 c4 c5 C6 c7 C8 c9 c10 c11 < - T11 T12 T13 T14 T15 T16 T17 ><-><-><->< ><-> <-> <-><-><--> <- For peptide T13, NTAVK and NTAVR were isolated.…”

Section: Molecular Massmentioning

confidence: 99%

Purification and Characterization of Tilapia (Oreochromis mossambicus) Deoxyribonuclease I

Hsiao

Wang

et al. 1997

DNase I of tilapia (Oreochromis rnossambicus) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg" as activator. The CaZ+/Mg2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55 "C, but is not inactivated by trypsin or 2-mercaptoethanol under alkaline conditions, with or without CaCI,. Its isoelectric point is 6.0. The 258-amino-acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys+Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar-chain length. [5]. The enzymatic properties of DNase I in these three species are very similar, and their primary structures are highly conserved [6]. It has been reported 171 that although the enzymatic properties of shrimp DNase I are very similar to those of mammalian DNase I, the protein structure is very different. Due to its high content of disulfide bonds and a highly cross-linked structure, shrimp DNase I is relatively stable to heat or SDS treatment [8]. A DNase-I-like enzyme, which is inhibited by muscle and liver actin, has been isolated from carp liver [9]. We investigated fish DWase 1 because fish are vertebrates but have a fused hepatopancreas similarly to shrimp. RNA was isolated from a piece of fresh small intestine of the fish with TRIzol Reagent (Life Technologies). Iodoacetic acid and P-mercaptoethanol were obtained from Wako. DEAE-cellulose (DE-52) was from Whatman. Hydroxyapatite (DNA grade) was from Bio-Rad. Trypsin and phenyl-Sepharose 4B were from Sigma. Sephadex G-I 00, ampholytes and concanavalin-A-Sepharose were from Pharmacia. Purified bovine DNase I was obtained via the method described previously [lo]. MATERIALS AND METHODS MaterialsDNase I assay. DNase I activity was determined by the hyperchromicity method [3, 101. 1 U enzyme activity causes an increase in absorbance of 1 at 260 nm in a 1-cm cuvette, per min per ml of assay mixture at 25°C.Purification of tilapia DNase 1. Approximately 450 g of tilapia hepatopancreas was minced and homogenized with a homogenizer (Heidolph DIAX 600) in 400ml 20mM TrislHCl, pH 8.0. Cell debris were removed by centrifugation in a Beckman JA14 rotor at 15 000 rpm for 1 h. The supernatant was filtered through two layers of cheese cloth, and the clear solution, termed crude extract, was subjected to (NH,),SO, fractionation between 35 % and 65 70 saturation. The precipitate obtained was dissolved in SO ml 20 mM Tris/HCl, pH 8.0, and dialyzed against the same buffer for 12 h. The dialyzed solution was passed through a DEAE-cellulose column (5 cmX15 cm), and the column was washed with 900ml of the same buffer. The absorbed proteins were eluted with a linear gradient of NaCl from 0 to 0.2 M at a flow rate of 60 ml/h. DNase I was eluted in approximately 0.05 M ...