Annie Delpech

Localization of Hyaluronectin in the nervous system

The localization of hyaluronectin was determined by immunofluorescence and immunoperoxidase methods, in the rat, the sheep and the human. The study of the peripheral nervous system revealed the localization of this protein at the node of Ranvier. It was also present at this site in the central nervous system where the appearance was less characteristic than in the peripheral nervous system. The protein was also observed around about 10% of neurones in all of the regions studied. The subcellular structures labelled could not be precisely defined with the optical microscope.

Expression of hyaluronic acid-binding glycoprotein, hyaluronectin, in the developing rat embryo ☆

Immunological and histological methods have been applied to the developing rat embryo to study the distribution of hyaluronectin (HN, a glycoprotein with hyaluronic acid-binding properties) previously shown to be present in the nervous system and in desmoplasias. HN was absent in the morula and the blastula and was first detected in the mesenchyme bordering the neural tube and somites on Day 10, i.e., at a time when hyaluronic acid is already widely dispersed in the mesenchyme. At this stage HN appeared to be closely associated with the basement membrane around the epithelial structures (somites, notochord, ectoderm) whereas the intercellular areas of mesenchyme were less strongly strained. The delineation of basement membranes decreased progressively, while the accumulation of HN increased in the cell-free areas of mesenchyme, giving a continuous, diffuse pattern. Differentiation of mesenchyme into vertebral cartilage and gut smooth muscle was accompanied by a progressive disappearance of HN. Even after streptomyces hyaluronidase or chondroitinase digestion the antigen was not unmasked in these tissues. The results are in agreement with the few observations made in the human. They suggest that HN could play a role, in association with fibronectin and glycosaminoglycans (hyaluronic acid), in the physiology of the embryonic extracellular matrix. HN appeared at a later stage in the embryonic nervous tissue; its distribution was extracellular in areas where both cell migration and proliferation occur.

Characterization of hyaluronic acid on tissue sections with hyaluronectin.

An affinity immunological procedure for hyaluronic acid detection on tissue sections is described. This new, sensitive, and specific technique is based on the high affinity of hyaluronectin for hyaluronic acid, utilizing anti-hyaluronectin-hyaluronectin immune complexes. Elimination of binding when the reagent was supplemented with hyaluronic acid or when Streptomyces hyaluronidase-digested tissue sections were used emphasizes the specificity of the assay. This technique made possible accurate HA localization in embryonic mesenchyme, in neural tissue, in kidney medulla, and in tumors.

The origin of hyaluronectin in human tumors.

The origin of tumor stroma hyaluronectin (HN), a glycoprotein that binds to hyaluronan (HA), has long remained unknown. Histological observations of human tumors suggest that tumor HN could originate from stroma fibroblasts, and in some cases from inflammatory cells. The fibroblast origin was confirmed by the discovery of HN‐like antigen along with hyaluronan in culture medium of tumor‐derived fibroblasts. An HA‐binding protein was characterized in the culture medium of peripheral blood mononuclear cells (PBMC) in both normal subjects and tumor‐bearing patients and was found to be human HN. Cultivated monocytes did not produce HA. HN was not related to the HA‐binding site CD44. Sequencing of brain HN‐derived peptides demonstrated that each determined peptide sequence was similar to a sequence of the proteoglycan PG‐M/versican, suggesting that HN is the HA‐binding moiety of the proteoglycan. One probe was synthesized from human PBMC by polymerase chain reaction with primers derived from HN sequences also found in versican. Northern blots were positive only with HN‐producing cells. The main RNAs were in the 6–8 kb range, and there was a limited proportion of smaller RNA, which was compatible with the size expected from the HN molecular mass. Southern blotting of monocytes and tumor cells demonstrated that the gene was limited to a unique band. We conclude that HN, an extracellular component of brain, connective embryonic, inflammatory and tumoral tissues, is a PG‐M/versican‐derived molecule. Our results suggest that tumor HN, which originates from fibroblasts and monocytes of tumor stroma, is a molecular component of the host‐tumor relationship and could play a role in the regulation of HA activity in oncogenesis. Int. J. Cancer 72:942–948, 1997.

Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin.

1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. The KD for high molecular weight HA was 5.4 10(-9) M at 37 degrees C and lower than 10(-10) M at 4 degrees C. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. The specific site for interaction was the HA-derived decasaccharide HA10. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage.

Hyaluronectin in normal human skin and in basal cell carcinoma

The localization of hyaluronectin has been studied in normal skin and in basal cell carcinoma. In fetal skin it is abundant in the dermis but absent from the epidermis, and in adult skin it is totally absent except in the hair sheaths and bulbs. In basal cell carcinoma it is abundant only in the stroma reaction. The presence of this protein in mesenchymatous tissues seems to be linked to zones of physiological or neoplastic proliferation.

Brain Glycoprotein in Tumours of the Nervous System

We studied various tumours of the nervous system by the immunofluorescence technique using an anti-brain specific alpha 2 glycoprotein antiserum (anti-NSA3 antiserum). We found the antigen in 24/27 astrocytomas and 4/4 oligodendrogliomas but in none of the 8 meningiomas tested. There was an identity between the astrocytoma/oligodendroglioma antigen and that of normal brain as shown by the immunoprecipitation technique. By the immuno-fluorescence technique using inhibition of the antiserum we demonstrated that the tumour antigen is devoid of some specific nervous system determinants present in normal brain.

Specificity of hyaluronectin binding to hyaluronic acid.

In a recent report, Ripellino et al. (1) presented a nice technique for detection of tissue hyaluronic acid. The technique, like that already published by Knudson and Toole (2), is based on the specific affinity ofa protcoglycan for hyaluronic acid. In the last eight lines of their article, Ripellino et al. criticize a work presented earlier by our group in which we used hyaluronectin, another hyalunonic acid-binding glycopnotein, to detect tissue hyaluronic acid. They stated: “The specificity and binding properties of hyalunonectin . . . have not yet been cleanly established.” We do not believe this statement is objective. According to the paper of Ripellino et al., the specificity criteria for binding of the protcoglycan-link protein complex to hyalunonic acid arc (a) blocking of binding when the complex is pre-incubated with oligosacchanides of specific size (10-18 or 16-30 monosaccharides), (b) absence of blocking by chondroitin sulfate, and (c) suppression of binding by pre-incubation of the tissue 5cction with Streptomyces hyalunonidase. These three conditions were fulfilled in the hyalunonectin study using both biochemical tests and tissue sections. Moreover, we could observe (3,4,5) that other glycosaminoglycans (chondroitin sulfates A, B, or C-kenatan sulfate-hepanin) did not interfere even in the very sensitive ELISA test. The blocking effect ofhyalunonic acid in the enzymo-immunological assay (6) or on tissue sections (7) was not only obtained with mixtures ofoligosacchanidcs but with purified HAlO, HA12, and HA14 hyalunonic acid-derived oligosacchanides, and was compared with the effect of smaller oligosaccharides HA9, HA8, HA6, and HA4. The effect of high molecular weight hyaluronic acid was tested by chromatography, by acnylamide gel electnophoresis, by immunodiffusion tests, and with the enzymo-immunological assay. Hyaluronic acid was found to be the only GAG to interfere with hyalunonectin behavior. The authors also based their reservations on the fact that “the staining pattern observed by these investigators is somewhat different from that found in our studies.” We cannot say as far as electron microscopy is concerned, but the optical microscopic pattern published by Ripellino et al. seems to us very similar to the pictures obtained with our technique (7) for hyaluronic acid (and not for hyaluronectin, which gives less extended staining). Both techniques probably have their own advantages. Both need the same type of controls, of which the most important is the blocking effect of HAlO vs. the absence of blocking by HA8. Both, however, encounter the same pitfall, not mentioned by the authors, which is that hyalunonic acid may be obliterated (saturated) by tissue components that have affinity for it, which could lead to false negative results.