Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

1993-06

AUTHORS

Jingsong Wang, Chih-Hao Chou, Joel Blankson, Minoru Satoh, Mark W. Knuth, Robert A. Eisenberg, David S. Pisetsky, Westley H. Reeves

ABSTRACT

The Ku autoantigen is a DNA binding factor consisting of 70 and ∼80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factorsin vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of ∼1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610–705, 8–221, and 1–374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506–541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immunological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku. More... »

PAGES

15-28

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/bf01006891

DOI

http://dx.doi.org/10.1007/bf01006891

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1038786216

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/7694076


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31 schema:description The Ku autoantigen is a DNA binding factor consisting of 70 and ∼80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factorsin vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of ∼1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610–705, 8–221, and 1–374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506–541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immunological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku.
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38 schema:keywords DNA
39 DNA-dependent protein kinase
40 Ku
41 Ku autoantigen
42 Ku fusion proteins
43 Ku monoclonal antibodies
44 Ku protein
45 L-929
46 L-929 cells
47 MAbs N3H10
48 MOPC-315
49 MRL/lpr mice
50 N3H10
51 N9C1
52 S10B1
53 S5C11
54 abundance
55 addition
56 amount
57 analysis
58 antibodies
59 antigenic determinants
60 assembly
61 autoantibodies
62 autoantigens
63 autoimmune sera
64 cell lysates
65 cells
66 cellular Ku
67 certain transcription factorsin
68 characterization
69 comparable reactivity
70 defined regions
71 determinants
72 dimer
73 distinct epitopes
74 distribution
75 epitopes
76 factors
77 factorsin
78 function
79 function of Ku
80 fusion protein
81 heterodimers
82 human autoimmune sera
83 human p80
84 humans
85 immune response
86 immunoblot
87 immunoblot analysis
88 immunofluorescence studies
89 immunological reagents
90 kinase
91 lack
92 lack of antibodies
93 level of Ku
94 levels
95 low abundance
96 lpr mice
97 lysates
98 mAbs
99 mAbs 111
100 mice
101 monoclonal antibodies
102 most non-primate cells
103 most nonprimate cells
104 most patient sera
105 murine Ku
106 murine autoantibodies
107 murine monoclonal antibodies
108 murine p70
109 murine p80
110 non-conserved antigenic determinants
111 non-primate Ku proteins
112 non-primate cells
113 nonprimate cells
114 observations
115 origin
116 p70
117 p70/
118 p80
119 panel
120 patient sera
121 polyclonal immune response
122 presence
123 present study
124 previous observations
125 previous studies
126 primate cells
127 primate origins
128 protein
129 protein kinase
130 reactivity
131 reagents
132 recombinant human
133 region
134 response
135 serum
136 site of p70
137 sites
138 small amount
139 specificity
140 study
141 suitability
142 transcription factorsin
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