The influence of the nature of the asparagine 289-linked oligosaccharide on the activation by urokinase and lysine binding properties of natural and recombinant human plasminogens.

Davidson, Donald J.

doi:10.1172/jci116557

Cited by 19 publications

(6 citation statements)

References 24 publications

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“…However, despite the fact that the nature of the glycan assembled on Asn289 of Pg does influence somewhat the binding strength of ai-amino acids to this protein (Davidson & Castellino, 1993), these small differences do not affect the major conclusions of this investigation.…”

Section: Discussionmentioning

confidence: 85%

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

Menhart

Hoover²,

McCance³

1995

Biochemistry

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In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 85%

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

Menhart

Hoover²,

McCance³

1995

Biochemistry

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“…Some studies have shown that the N-linked glycans on recombinant proteins secreted from insect cells are important for functional properties. For example, ligand binding of the thromboxane A 2 receptor (72), secretion of decorin (62), and the ability of human plasminogen to be activated by urokinase (73) are all influenced by N-glycan substitution. At present we do not know whether differences in Nlinked glycosylation may contribute to the inability of atrolysin C to cleave the Asn 341 2 Phe bond in rG1-G2.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase

Mercuri

Doege

Arner

et al. 1999

Journal of Biological Chemistry

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A recombinant human aggrecan G1-G2 fragment comprising amino acids Val 1 -Arg 656 has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M r 90,000 -95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. sequences were used to confirm aggrecanase cleavage at the Glu 373 2 Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1␣ or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.Aggrecan is a chondroitin sulfate and keratan sulfate-bearing proteoglycan. It is present in cartilage as large multimolecular aggregates bound noncovalently to hyaluronan (HA). 1Aggregate formation is mediated via the proteoglycan tandem repeat loops in the N-terminal G1 domain which bind to decasaccharide units within the polymeric HA. This binding is further stabilized by link protein, which binds to form stable trimeric complexes. The formation of large aggregate structures provides a mechanism for trapping aggrecan in the tissue and preventing its loss by diffusion. A major feature of the pathology of arthritis is the gradual loss of aggrecan from the cartilage matrix. This loss involves proteolysis of the core protein and release into culture medium (1, 2) or synovial fluid (3) of large fragments lacking G1 domains that are t...

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“…Indeed, the original extensive survey by Warren using the thiobarbituric acid test failed to show a positive response in a wide variety of protostomes examined . Furthermore, with one possible exception, recombinant proteins expressed in insect cells are not reported to have sialic acids. , However, recent studies have shown that some insects may express Neu5Ac in a stage-specific manner. , Also, octopuses and squids (belonging to the molluscs) have recently been shown to express gangliosides (sialic acid-containing glycolipids) . Perhaps sialic acids are expressed only in a small number of protostome species.…”

Section: A Eukaryotes1 Animalsmentioning

confidence: 99%

Chemical Diversity in the Sialic Acids and Related α-Keto Acids: An Evolutionary Perspective

2002

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The influence of the nature of the asparagine 289-linked oligosaccharide on the activation by urokinase and lysine binding properties of natural and recombinant human plasminogens.

Cited by 19 publications

References 24 publications

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase

Chemical Diversity in the Sialic Acids and Related α-Keto Acids: An Evolutionary Perspective

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