Abstract
The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.
Highlights
The complete amino acid sequence of the Fab frag,cytomegalovirus, and ment ofprotein KAU, a human monoclonalcold agglutinin (IgMk) with anti-I activity,was determined
Recent serological studies and partial amino acid sequence analysis show that these autoantibodies are composed by light chain (L-chain) belonging to the Vklllb sub-subgroup and H-chains thatderive from theVHIV subgroup (8)
The Fab fragment of the isolated protein was generated by limited trypsin digestion and separated from intact IgM and (Fc)Sp fragmenton Sepharose CL-GB as indicated in Fig. 1.The L-chain and the Fd fragment composing KAU Fab were successfully isolated via HPLC, using partial reduction and alkylation of the Fab fragment, As indicated in Fig. 2, discontinuous gradient at 53% buffer B (0.1%trifluoroacetic acid, 66%acetonitrile) yielded two peaks corresponding to the L-chain
Summary
Trypsin-Five milligrams of completely reduced and "C-alkylated KAU light chain were dissolved in 1 ml of 0.2 M NH,HC03, pH 8.0, and digested for 20 min at 37 "C with TPCK-treated trypsin a t a 1:lOO (w/w) enzyme/protein ratio. Endoproteinase Lys-C-One milligram of completely reduced and "C-alkylated Fd was dissolved in 25 mM Tris-HCI, 1 mM EDTA, pH 8.5, and digested with endoproteinase Lys-C (Boehringer Mannheim) a t a 1:lOO (w/w) ratio for 18 h a t 37 "C. Endoproteinase Asp-N-One hundred micrograms of peptide K6 (obtained from endoproteinase Lys-C digestion of the Fd fragment) and 1 mgof completely reduced and "C-alkylated L-chain were dissolved in 50 mM phosphate buffer, pH 8.0, and proteolytically cleaved with endoproteinase Asp-N (Boehringer Mannheim) a t a 1:lOO ratio (w/w) for 18 h a t 37 "C. The carboxyl-terminal amino acids released weredetermined by amino acid analysis without hydrolysis using a Picotag amino acid analyzer (Waters Associates)
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