Ribonuclease-Gold Labels Heparin in Human Mast Cell Granules: New Use for an Ultrastructural Enzyme Affinity Technique

Abstract

We evaluated an enzyme affinity-gold ultrastructural technique designed to identify RNA-rich structures, based on an RNase-gold (R-G) probe in human mast cells (HMCs). As expected, the R-G technique labeled RNA-containing ribosomes and nucleoli in HMCs. The heparin-rich secretory granules in HMCs were also labeled. Extensive studies revealed that HMCs isolated from lung or skin and sustained in short-term cultures, derived de novo in growth factor-supplemented cord blood cell cultures, or present in vivo in multiple sites all shared this property. We performed a large number of controls designed to examine the HMC granule binding characteristics of gold alone, of irrelevant protein- or enzyme-gold reagents, of the role of charge and enzyme activity after various enzyme digestions, after blocking with macromolecules, after exposure to inhibitors of RNase, of heparin, or to irrelevant enzyme inhibitors, including staining of macromolecule-containing test agar blocks and a variety of combined absorption and digestion experiments of the binding of R-G to HMC granules. These studies established that the R-G method detected heparin in this site in conventionally prepared, well-preserved electron microscopic samples. These findings demonstrate a new use for this enzyme affinity-gold technique in mast cell biology, based on the known property of heparin as an inhibitor of RNase.

Localization of DNA and RNA in Eosinophil Secretory Granules

“…RNase digestion of sections prior to immunogold labeling resulted in a marked reduction in the immunogold labeling in nuclei and granules, indicating that the labeling, for the most part, represents RNA. The failure to completely remove RNA with ribonuclease is consistent with previous studies [15,44]. Similarly, in human eosinophils, use of DNase I and RNase prior to DNA immunostaining and RNA staining with molecular probe resulted in marked reduction in DNA and RNA fluorescent signals in both nuclei and granule compartments, suggesting the specificity of our staining procedures.…”

Ultrastructure of Human Mast Cells

“…3). All of these morphological observations informed our pursuit of a possible synthetic role for HMC cytoplasmic granules [24, 25, 26, 27, 28, 29, 30]. These recently published studies in aggregate suggest that HMC granules are equipped for such a role, and they considerably augment our vision of the role of secretory-storage granules in the cell biology of all granulated secretory cells.…”

“…Where morphological reviews are available, they are cited without extensive recapitulation. Rather, we draw attention to newer studies of HMC organelles and their possible functional relationships to eicosanoid [16, 17], cytokine [18, 19, 20, 21, 22, 23], and RNA biology [24, 25, 26, 27, 28, 29, 30]. …”

“…Ultrastructural immunocytochemical and enzyme-affinity labeling techniques have documented storage of proteases, such as tryptase and chymase [33], a mediator of inflammation, such as histamine [9, 10], and the proteoglycan, heparin [24, 25, 34]in HMC granules. HMCs have also been shown to produce a variety of cytokines.…”

Proteoglycans support proper granule formation in pancreatic acinar cells

“…In addition, well-known constituents of ZG such as RNAse A and carboxyl ester lipase (CEL) have the potential to interact with proteoglycans, e.g. to influence enzyme function (Dvorak & Morgan 1998, Rebaï et al 2005. Moreover, an interaction of the secretory lectin ZG16p (Cronshagen et al 1994, Kleene et al 1999) with heparan sulfate containing proteoglycans from ZG has recently been reported (Kumazawa-Inoue et al 2012).…”