Mass Determination, Subunit Organization and Control of Oligomerization States of Keyhole Limpet Hemocyanin (KLH)

Abstract

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH ; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLHl and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLHl subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45 -60 kDa functional domains (termed l a through lh) and determined their sequential arrangement. The KLHl domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain lh, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLHl and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional l h domain. It was also shown that the KLHl didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Triskaline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl, and 5 mM MgCI,, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLHl multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

The Sequence of a Gastropod Hemocyanin (HtH1 from Haliotis tuberculata)

“…This is very close to the value of ϳ400 kDa measured in SDSpolyacrylamide gel electrophoresis for both HtH1 and KLH1 (12,24). From the multitude of conserved and variable structural features visible in Fig.…”

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1‐h) reveals disulfide bridges

“…Briefly, KLH1-h was prepared from purified KLH1 by limited proteolysis with V8 protease and subsequently purified by ion exchange chromatography (20,26). KLH1-h was then crystallized at pH 4.0 in sodium citrate buffer in the presence of 9.0% PEG 1000 and 50-mM K 2 HPO 4 .…”

Keyhole Limpet Hemocyanin: Molecular Structure of a Potent Marine Immunoactivator

“…On the other hand, if purified and stable oligomers are subjected to analytical ultracentrifugation, accurate sedimentation coefficient and molecular weight data are likely to be obtained. Mass determination obtained from unstained dark-field images of KLH1 and KLH2 (subunits and didecamers) by scanning transmission electron microscopy (STEM) [14]provided quantitative data which correlate well with the sedimentation coefficient values, and at the same time confirm the presence of several oligomeric forms. Light scat…”

“…390 kD for KLH1 vs. approx. 360 kD for KLH2 [14]). Preparative native electrophoresis has been used to purify the two subunits [6, 15], but most efforts have been directed towards the purification of the intact molecules.…”