Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.     

Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits.

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the alpha- and beta-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of alpha-subunit linked to a 54-amino-acid endopeptide and beta-subunit was lower than with the subunits alone.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Expression in non-lymphoid cells of mouse recombinant immunoglobulin directed against the tumour marker human placental alkaline phosphatase

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.     

A possible difference in the heterogeneity of the heavy and light chains from a rabbit anti-p-azobenzoate antibody preparation.

CNBr cleavage of rabbit heavy (H) chains leads to the formation of a fragment, C-1, which consists of the N-terminal half of the H chain. Fragment C-1 is cleaved at methionyl residues but held together by intrachain S-S bonds so that smaller fragments can be liberated by total reduction and alkylation. In the case of the C-1 fragment from an anti-p-azobenzoate antibody preparation, which has a light (L) chain of markedly restricted heterogeneity, total reduction and alkylation liberated seven major fragments in good yield. The N-terminus of two of these fragments corresponds to position 35 of the H chain but their N-terminal sequences are clearly different. The H chain regions represented by the other fragments implied that they were derived from H chains having different distributions of methionyl residues. This hypothesis was supported by isolating six different antibody components from the antibody preparation by isoelectric focusing and then digesting them with CNBr. Comparison of the products showed that the six components all appeared to behave differently. These results are interpreted as suggesting that the process whereby H and L chains are paired in vivo may not be completely specific and may provide a simple means of generating a significant contribution to antibody diversity.     

Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry

ABSTRACT

The analysis of monoclonal antibodies (mAbs) by a middle-down mass spectrometry (MS) approach is a growing field that attracts the attention of many researchers and biopharmaceutical companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before MS analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light and heavy chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ~24 min, which is 40-60% faster than regular liquid chromatography-MS/MS analysis, to acquire fragmentation data using different dissociation methods.     

Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens

Chain recombination experiments with a set of structurally and/or functionally related antibodies were performed to assess the role of the heavy (H) and light (L) chains in determining antigen specificity. The results demonstrated that specificity for hen egg white lysozyme vs two haptens (dinitrophenyl or galactan) is H chain determined and for one set of proteins could be attributed specifically to the H3 region. In contrast to hapten vs lysozyme specificity, when reassociated molecules derived from structurally unrelated antibodies that bound nonoverlapping epitopes on lysozyme were tested, localization of binding to a particular epitope on lysozyme could be predominated by either H or L chains. Furthermore, in some cases, unique specificities distinct from those of either parental antibody were formed. Replacement of the native L chain with an isotypically homologous L chain was more likely to restore high affinity protein binding than was replacement of a less related L chain. When isotypically homologous L chains were compared in association with the same H chain, fine specificity profiles were sensitive to substitutions in as few as two residues that could be attributed to somatic mutation. These results demonstrate that both affinity and specificity derive from very subtle interactions between H and L chains and provide examples of how VH assembly, VL-VH pairing, and somatic mutation could contribute to development and maturation of the specificity repertoire.     

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed.     

Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production

Monoclonal antibodies and antibody fragments are used for diverse diagnostic and therapeutic applications. We have investigated the secretory production of Fab fragments from insect cells cotransfected with plasmid vectors carrying heavy- and light-chain genes. In the present study, to promote the formation of the disulfide bond between the heavy and light chains, some positively charged amino acid residues were introduced near the cysteine residue for the disulfide bond at the C-terminus of C_L, while some negatively charged amino acid residues were added near the cysteine residue for the disulfide bond at the C-terminus of C_H1. This electrostatic steering led to an increase in Fab secretions from insect cells.     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.     

Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.

Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.