Monoclonal antibodies specific for the C-terminus of the laminin B2 subunit. Evidence for glycosylation differences between murine and human laminin.
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ABSTRACT:
SUBMITTER: Brown JC
PROVIDER: S-EPMC1151281 | biostudies-other | 1991 Aug
REPOSITORIES: biostudies-other
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Project description:Transcriptional regulation of Laminin expression during embryogenesis is a key step required for proper ECM assembly. We show, that in Drosophila the Laminin B1 and Laminin B2 genes share expression patterns in mesodermal cells as well as in endodermal and ectodermal gut primordia, yolk and amnioserosa. In the absence of the GATA transcription factor Serpent, the spatial extend of Laminin reporter gene expression was strongly limited, indicating that Laminin expression in many tissues depends on Serpent activity. We demonstrate a direct binding of Serpent to the intronic enhancers of Laminin B1 and Laminin B2. In addition, ectopically expressed Serpent activated enhancer elements of Laminin B1 and Laminin B2. Our results reveal Serpent as an important regulator of Laminin expression across tissues.
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Project description:Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain's thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2-8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.
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Project description:Around 70% of circulating alpha-2-antiplasmin (?2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-?2AP into the more potent fibrinolysis inhibitor Asn-?2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-?2AP in a purified system, with ~8-fold faster conversion of Met(R6)-?2AP than Met(W6)-?2AP. To date, assays to determine N-terminally intact Met-?2AP in plasma have been limited to an ELISA that only measures Met(R6)-?2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-?2AP, Met(W6)-?2AP and all ?2AP forms (total-?2AP) in order to develop specific Met(R6)-?2AP and Met(W6)-?2AP ELISAs. Recombinant Met(R6)-?2AP, Met(W6)-?2AP and Asn-?2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-?2AP and Met(W6)-?2AP. All mAbs were evaluated for their specific reactivity using the three recombinant ?2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-?2AP and Met(W6)-?2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-?2AP and used for detection. With the novel ELISAs we determined Met(R6)-?2AP and Met(W6)-?2AP levels in plasma samples and we showed that Met(R6)-?2AP was converted faster into Asn-?2AP than Met(W6)-?2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-?2AP and Met(W6)-?2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of ?2AP in plasma samples.
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