Lymphocyte membrane immunoglobulins: similarities between human IgD and mouse IgD‐like molecules

Abstract

131I‐labeled IgD‐like molecules extracted from the membrane of mouse spleen, and lymph node cells and their constitutive chains have been characterized by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS‐PAGE) followed by radioautography. Unreduced mouse IgD‐like molecules concentrated in two definite bands (IgD1 and IgD2). IgD, migrated slightly ahead of 131I‐labeled mouse membrane IgM and displayed a mobility very similar to that of 131I‐labeled IgD extracted from the membrane of human tonsil cells. IgD2 migrated faster than both IgD1 and human membrane IgD (mIgD), but slower than 14C‐labeled mouse IgG2 b used as marker. The different mobility of IgD1 and IgD2 was due to difference in size of their respective H‐chains (δ1 and δ2), as shown by SDS‐PAGE analysis of the two molecules after reduction. Mol.wt. determination of IgD1, IgD2 and of the corresponding constitutive chains are consistent with an H2L2 structure. IgD1 and IgD2 were partially replaced following prolonged dialysis by molecules with a SDS‐PAGE mobility consistent with an HL structure, IgD3. In contrast to that found for spleen and lymph node, IgD1 only was detected on the surface of bone marrow cells. Immunofluorescence experiments have shown that while the majority of mouse lymph node or spleen cells bear IgM and IgD, IgM was found to be the predominant Ig class on the surface of bone marrow cells, although a certain number of IgD‐IgM and a few IgD‐positive cells were found. Experiments where mouse lymphocytes have been treated with pronase have indicated that, in analogy with mlgD of primates, mouse IgD‐like molecules are very labile and can be removed from the cell surface by very mild proteolytic treatment.

Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

“…Previous studies analyzing 8 chains from mixed populations ofcells or immunoglobulins have identified heterogeneity in the NaDodSO4 gel electrophoresis profiles of 8m and 8s (9,10,(13)(14)(15)(16)(17)(18)(19). This heterogeneity may be related to the observation, in the present investigation, of differential N-glycosylation of the SeD 8 chains.…”

“…As shown by charge-shift electrophoresis, these forms probably have a hydrophobic COOH terminus (11,12). Studies using one-dimensional NaDodSO4 gel electrophoresis systems have demonstrated that am exists as two molecular entities (8im and 8m2), distinguished from one another by a difference in molecular weight (ranging from 2000 to 7000) (13)(14)(15)(16). Likewise, 8, has been resolved into two species (5s1 and 8s2) that differ in molecular weight (17)(18)(19).…”

Presence of serum IgD and IgD‐containing plasma cells in the mouse

“…Briefly, the Nonidet-P40 lysate of spleen and lymph node cell suspensions from 300 BALBk mice was depleted of IgG, IgM and IgA by immunoabsorption with the corresponding specific antisera coupled to Sepharose 4 B [9]. The lysate was finally applied to a Sepharose anti-F(ab'), column, the bound material eluted with 10 M urea, dialyzed against phosphate-buffered saline (PBS) and repeatedly injected into a rabbit.…”

“…The lysate was finally applied to a Sepharose anti-F(ab'), column, the bound material eluted with 10 M urea, dialyzed against phosphate-buffered saline (PBS) and repeatedly injected into a rabbit. The antiserum was absorbed with mouse red blood cells, a Sepharose-bound lysate from mouse T cell lymphoma E L 4 [lo] and Sepharose-bound IgM, IgA, IgG (x and h), prepared from various myelomas, as previously described [9]. After such procedures, the antiserum did not react with 14C-labeled Ig proteins secreted by mouse myeloma Rabbit anti-mouse a, p, y and L chain (having both anti-x.…”

“…After such procedures, the antiserum did not react with 14C-labeled Ig proteins secreted by mouse myeloma Rabbit anti-mouse a, p, y and L chain (having both anti-x. and anti-h activity) and anti-phytohemagglutinin (PHA) antisera were prepared as described in detail previously [9]. Aliquots of all of the above reagents were insolubilized on Sepharose 4 B [9].…”