Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-06-09
2003-11-25
Jones, W. Gary (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S023600, C800S278000, C800S295000, C435S006120, C435S320100, C435S410000
Reexamination Certificate
active
06653459
ABSTRACT:
The present invention relates to plant GnTI sequences, in particular, plant nucleic acid sequences encoding the enzyme N-acetyl glucosaminyl transferase I (GnTI), as well as GntI antisense or sense constructs, deduced therefrom, and their translation products, antibodies directed against said translation products as well as the use of the sequence information for the production of transformed microorganisms and of transgenic plants, including those with reduced or lacking N-acetyl glucosaminyl transferase I activity. Such plants with reduced or lacking N-acetyl glucosaminyl transferase I activity are of great importance for the production of glyco-proteins of specific constitution with respect to their sugar residues.
PRIOR ART
In eukaryotes, glycoproteins are cotranslationally assembled in the endoplasmatic reticulum (ER) (i.e. during import into the ER lumen) by the attachment of initially membrane bound glycans (via dolichol pyrophosphate) to specific asparagine residues in the growing polypeptide chain (N-glycosylation). In higher organisms, sugar units located at the surface of the folded polypeptide chain are subjected to further trimming and modification reactions (ref. 1) in the Golgi cisternae. Initially, typical basic Glc
3
Man
9
GlcNAc
2
units of the high-mannose type are formed by means of different glycosidases and glycosyl transferases in the ER, which during the passage through the different Golgi cisternae are subsequently converted to so-called complex glycans. The latter are characterized by less mannose units and the presence of additional sugar residues, such as fucose, galactose and/or xylose in plants and sialic acid (N-acetyl neuraminic acid, NeuNAc) in mammals (ref. 1,2,3). The extent of the modifications can differ between glycoproteins. Single polypetide chains may carry heterogeneous sugar chains. Furthermore, the glycosylation pattern may vary for a specific polypeptide (tissue specific differences), and does not always have to be uniform with respect to a specific glycosylation site, which is referred to as microheterogeneity (ref. 4,5). Up to now, the role of asparagine bound glycans is barely understood, which i.a. results from the fact, that said glycans may serve several functions (ref. 6). However, it can be assumed, that e.g. protection of a polypeptide chain from proteolytic degradation can also be achieved by glycans of a more simple oligomannosyl type (ref.7).
DESCRIPTION OF PROBLEMS
Glycoproteins are highly important in medicine and research. However, large scale isolation of glycoproteins is time-consuming and expensive. The direct use of glycoproteins isolated conventionally often raises problems, since upon administration as a therapeutic, single residues of the glycan components may cause undesired side effects. In this context, the glycan component predominantly contributes to the physico-chemical properties (such as folding, stability and solubility) of the glycoproteins. Furthermore, isolated glycoproteins, as already mentioned above, rarely carry uniform sugar residues, which is referred to as microheterogeneity.
For the production of glycoproteins for medicine and research, yeasts prove to be unsuitable, since they are only able to perform glycosylations of the so-called high-mannose type. While insects and higher plants exhibit complex glycoprotein modifications, these, however, differ from those of animals. Therefore, glycoproteins isolated from plants have a strong antigenic effect in mammals. In most cases, animal organisms with glycosylation defects are not viable, since terminal glycan residues (e.g. of membraneous glycoproteins) mostly possess biological signal function and are indispensable for cell-cell-recognition during the course of embryogenesis. Mammalian cell lines with defined glycosylation defects already exist, the cultivation of which, however, is labour-intensive and expensive.
For mammals, different glycosylation mutants have been described in detail at the cell culture level (ref. 7,8,9,10). Said mutants are either defective in biosynthesis of mature oligosaccharide chains attached to dolichol pyrophosphate or in glycan processing or show alterations in their terminal sugar residues, respectively. Some of these cell lines exhibit a conditional-lethal phenotype or are defective in intracellular protein transport. The consequences of said defects for the intact organism are difficult to estimate. It has been observed, that a modification in the pattern of complex glycans on the cell surfaces of mammals is accompanied by the formation of tumours and metastases, although a functional relationship could not yet unambiguously be demonstrated (ref. 9). Therefore, in mammals glycosylation mutants are very rare. These defects, summarized under HEMPAS (Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum lysis test) (ref. 10,11), are based either on a deficiency in mannosidase II and/or low levels of the enzyme N-acetyl glucosaminyl transferase II (GnTII), and have strongly limiting effects on the viability of the mutated organism. GntI knock-out mice, in which the gene for GnTI has been destroyed, already die in utero of multiple developmental defects (personal communication, H. Schachter, Toronto).
Until recently, no comparable mutants were known for plants. By the use of an antiserum, which specifically recognizes complex modified glycan chains of plant glycoproteins and which predominantly is directed against the highly antigenic &bgr;1→2 linked xylose residues (ref. 12), the applicant was able to isolate several independant mutants from an EMS mutagenized F2 population of the genetic model plant
Arabidopsis thaliana,
which no longer showed complex glycoprotein modification (complex glycan, cgl mutants). After at least five back-crosses, each followed by intermittent selfings (to screen for the recessive defects), the glycoproteins were analyzed. These glycoproteins mainly carried glycans of the Man
5
GlcNAc
2
type, indicating a defect in N-acetyl glucosaminyl transferase I (GnTI) (ref. 8). Indeed, the Arabidopsis cgl mutants lacked GnTI activity (ref. 13), which normally catalyzes the first reaction in the synthetic pathway to complex modified sugar chains (ref. 1). However, according to observations so far, the viability of the mutated plants is not affected. In recent publications, plants are suggested as a putative source for the production of pharmaceutically relevant glycoproteins or vaccines (ref. 14,15). There however, it was overlooked, that glycoproteins isolated from plants may cause severe immune reactions in mammals, which up to now obstructed the production of heterologous glycoproteins in cultivated plants.
The applicant could demonstrate by way of example for the Arabidopsis cgl mutant, that plants can manage without complex modified glycoproteins to a great extent (ref. 13). Initially, secretory proteins are normally glycosylated in the ER of the mutant. In the Golgi apparatus of the cgl mutant, however, the oligomannosyl chains linked to the polypetide backbone via asparagine residues (N-glycosylation) then remain at the stage of Man
5
GlcNAc
2
residues, since N-acetyl glucosaminyl transferase I (GnTI) activity is missing (FIG.
1
). By this bio-synthesis block, the plant specific complex glycoprotein modification and in particular the attachment of &agr;1→3 fucose and &bgr;1→2 xylose residues is prevented, whereby the strong antigenic effect on the mammalian organism is absent. However, Arabidopsis as a herb only has little utilizable biomass. Therefore, for the large scale production of biotechnologically relevant glycoproteins these cgl plants are less suitable. As an alternative, cultivars, especially Solanaceae, such as potato, tobacco, tomato or pepper and furthermore alfalfa, canola, beets, soybean, lettuce, corn, rice and grain, with missing or highly reduced GnTI activity, would be ideal for the production of heterologous glycoproteins in plants. For this purpose, the methods of homology-dependent gene silencing would be applicable (ref. 16, 17).
Goldberg Jeanine
Jones W. Gary
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